Antisense modulation of PAZ/PIWI domain-containing protein expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of PAZ/PIWI domain-containing protein. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding PAZ/PIWI domain-containing protein. Methods of using these compounds for modulation of PAZ/PIWI domain-containing protein expression and for treatment of diseases associated with expression of PAZ/PIWI domain-containing protein are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of PAZ/PIWI domain-containing protein. Inparticular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodingPAZ/PIWI domain-containing protein. Such compounds have been shown tomodulate the expression of PAZ/PIWI domain-containing protein.

BACKGROUND OF THE INVENTION

[0002] In eukaryotes, protein synthesis involves a complex series ofprotein and nucleic acid interactions that lead to the assembly of an80S ribosomal nucleoprotein complex, comprising the methionyl initiatortRNA (Met-tRNA_(i)) base paired with the initiation codon of a messengerRNA, and result in translation of the mRNA into protein. This process oftranslation initiation has several linked stages that require theparticipation of numerous proteins known as eukaryotic translationinitiation factors (eIFs). The first stage involves the association ofeukaryotic translation initiation factor 2 (eIF-2), GTP nucleotide, andthe initiator Met-tRNA_(i) to form a ternary complex, which then bindsto the 40S ribosomal subunit to form a 43S preinitiation complex. In thesecond stage, the 43S preinitiation complex associates with other eIFsand with the mRNA, while the third stage involves movement of the 43Scomplex along the mRNA, scanning for the initiation codon, and formationof the 48S initiation complex when the anticodon of the initiatorMet-tRNA_(i) base pairs with the initiation codon. Finally, in thefourth stage, several factors dissociate from the 48S complex, allowingthe 60S ribosomal subunit to bind and form an 80S ribosome in atranslation-competent initiation complex (Pestova et al., Proc. Natl.Acad. Sci. U. S. A., 2001, 98, 7029-7036).

[0003] The gene encoding PAZ/PIWI domain-containing protein (also knownas hypothetical protein FLJ12765) has been noted to bear homology to theeukaryotic translation initiation factor EIF2C and Rattus norvegicusGERp95 mRNAs, members of a family of genes that encode proteins withPIWI and/or PAZ domains believed to be involved in post-transcriptionalgene silencing (PTGS). The eukaryotic translation initiation factor 2C,1 (EIF2C1) protein promotes the AUG-directed binding of the initiatorMet-tRNA_(i) to 40S ribosomes by stabilizing the formation of thetranslation initiation eIF-2/Met-tRNA_(i)/GTP ternary complex andpromoting guanine nucleotide exchange. The EIF2C1 gene is a member of anevolutionarily conserved multigene family in humans that includesseveral genes related to the Q99 gene exhibiting increased expression intumors harboring a mutation in the Wilms tumor suppressor gene WT1.Human EIF2C1 has significant homology to the ARGONAUTE1 (AGO1) geneimportant for proper development of leaves and cotyledons in Arabidopsisthaliana as well as to a gene, F48F7.1, identified in the course of theCaenorhabditis elegans genome sequencing project (Koesters et al.,Genomics, 1999, 61, 210-218). Based on its homology to theEIF2C1/AG01/Q99 family of proteins, the PAZ/PIWI domain-containingprotein may also be associated with human cancers.

[0004] The rat GERp95 (Golgi ER protein 95 kDa) protein was identifiedas a Golgi complex and/or endoplasmic reticulum (ER) membrane-associatedprotein. Numerous GERp95 homologues were noted to occur in a wide rangeof multicellular organisms, and in particular, GERp95 was noted to be93.5% identical to the protein encoded by the rabbit homolog of EIF2C1.A technique called double-stranded RNA-induced gene silencing, or RNAinterference (RNAi) was used to generate a GERp95-null phenotype in C.elegans and show that the C. elegans GERp95 orthologue is important formaturation of germ-line stem cells in the gonad of the nematode (Cikaluket al., Mol. Biol. Cell, 1999, 10, 3357-3372). Subsequently, the GERp95protein was observed to engage an Hsp90 molecular chaperone proteincomplex prior to its association with intracellular membranes. Hsp90 isan abundant chaperone protein with a limited number of substrate types,most of which are signaling proteins that include protein kinases,steroid hormone receptors, nitric-oxide synthases, and telomerase.Furthermore, the Hsp90 protein was required for the stability and Golgilocalization of GERp95 in NRK rat kidney cells. Gerp95 and relatedproteins were, therefore, predicted to comprise a new class of Hsp90substrates involved in novel signaling pathways (Tahbaz et al., J. Biol.Chem., 2001, 276, 43294-43299). Thus, a family of highly conservedsignal-transducing proteins has been defined and its members arepredicted to have roles in regulation of cellular differentiation,development, fertility and gametogenesis, and PTGS (Cikaluk et al., Mol.Biol. Cell, 1999, 10, 3357-3372; Tahbaz et al., J. Biol. Chem., 2001,276, 43294-43299).

[0005] The gene encoding PAZ/PIWI domain-containing protein appears tobe a member of this multigene family. Within this family, the proteinsPIWI (from Drosophila melanogaster), AGO1 and Zwille (from A. thaliana),were seminal members, and thus, the name PAZ domain was given to theregion of homology these proteins share. The PIWI domain has beendefined as a 300-amino acid domain that typically occurs C-terminal tothe PAZ domain of proteins that contain both domains. PAZ-domain-onlyand PIWI domain only proteins have also been discovered. The exactfunction of these domains remains unknown but the proteins containingthem are believed to be important in mechanisms of cell fatedetermination, development and gene silencing (Cerutti et al., TrendsBiochem. Sci., 2000, 25, 481-482). Interestingly, the processes ofPTGS/RNAi and development have been partially uncoupled in hypomorphicmutants of the AGO1 gene. PTGS was observed to be more sensitive thandevelopment to mutations in AGO1, indicating that some members of thePAZ/PIWI domain-containing protein family may have independent functionsin these two processes, or that expression levels can differentiallyaffect the two processes. Furthermore, it has been suggested thatmembers of the PAZ/PIWI domain-containing protein family may havepartially redundant or non-redundant functions (Morel et al., PlantCell, 2002, 14, 629-639).

[0006] In addition to being a potent technique used to generatephenocopies of the null phenotype in the nematode, RNAi is also anatural biological defense used by various organisms to prevent viralreplication and infection as well as to silence transposon hopping inthe germline. When double-stranded RNA (dsRNA) corresponding to thesense and antisense sequence of an endogenous mRNA is introduced into acell, it mediates sequence-specific genetic interference, and thecognate mRNA is degraded and the gene silenced (Bass, Cell, 2000, 101,235-238; Montgomery and Fire, Trends Genet., 1998, 14, 255-258). BecausePAZ/PIWI domain-containing protein is also homologous to rde-1 in C.elegans, and rde-1 mutants completely lack an interference response,PAZ/PIWI domain-containing proteins are implicated in the RNAi mechanism(Morel et al., Plant Cell, 2002, 14, 629-639).

[0007] Cellular compartmentalization by the Golgi and ER is believed toincrease the efficiencies of cellular processes by controlling thespatial and temporal interactions of proteins, nucleic acids, andlipids, and it is now clear from studies of EIF2C1 (GERp95) in C.elegans, that these two organelles are directly involved in processesthat affect cellular differentiation. Mistargeting and/or alteredexpression of intracellular membrane-associated proteins has been shownpreviously to have profound effects on cell growth, morphology, andtumorigenicity, and cellular defects in the Golgi or ER underlie thepathophysiology of many human diseases such as familialhypercholesterolemia, polycystic kidney disease, Tangier disease, cysticfibrosis, mucopolysaccharidosis types I, IV, and VII, progeroidsyndrome, and many others. Several alternative mechanisms have beenproposed for regulation of the activity and localization of the GERp95protein in rat cells. One possibility is that there are cell-specificisoforms of this protein. Postranslational modifications have also beenproposed as a means of regulating subcellular localization or activity.It has also been proposed that GERp95 may be displaced from thetranslation initiation complex by the interfering dsRNA, directlypreventing its activity in the translation of a target mRNA (Cikaluk etal., Mol. Biol. Cell, 1999, 10, 3357-3372). Based on its structuralhomology, PAZ/PIWI domain-containing protein may be similarly regulatedby the existence of cell-specific isoforms, splice variants,posttranslational modifications or compartmentalization and havedifferent activities, possibly involved in the control of mRNAtranslation and/or RNAi.

[0008] Thus, PAZ/PIWI domain-containing protein is a potentialtherapeutic target in conditions involving aberrant function oftranslation initiation complexes or leading to altered gene expression,as well as in conditions resulting from mistargeting,compartmentalization, or misregulation of signaling pathways by thePAZ/PIWI domain-containing protein and dysregulation of stem celldifferentiation. PAZ/PIWI domain-containing protein is also a targetpotentially allowing for further elucidation of the mechanisms of RNAi.

[0009] Disclosed and claimed in PCT Publication WO 01/02568 is a libraryof polynucleotides, the library comprising the sequence information ofat least one of a group of nucleotide sequences, wherein one member ofsaid group bears a region of identity to 370 nucleotides of the geneencoding PAZ/PIWI domain-containing protein (nucleotides 1270-1639 ofGenBank Accession NM_(—)024852.1). Further claimed is an isolatedpolynucleotide comprising a nucleotide sequence having at least 90%sequence identity to an identifying sequence of said group or adegenerate variant or fragment thereof, a recombinant host cellcontaining the polynucleotide, an isolated polypeptide encoded by thepolynucleotide, an antibody that specifically binds said polypeptide,and a vector comprising said polynucleotide. Antisense oligonucleotidesare generally disclosed (Williams et al., 2001).

[0010] Disclosed and claimed in PCT Publication WO 01/77164 is a nucleicacid comprising a sequence at least 18 bases in length of a segment ofthe chemically pretreated DNA of genes associated with metabolism takenfrom a group of sequences in which a member of said group bears a regionof identity to 20 nucleotides of the gene encoding PAZ/PIWIdomain-containing protein (nucleotides 1491-1510 of GenBank AccessionNM_(—)024852.1) as well as sequences complementary thereto. Furtherclaimed is an oligonucleotide or peptide nucleic acid (PNA)-oligomercomprising a sequence having a length of at least 9 nucleotides whichhybridizes to or is identical to a chemically pretreated DNA of saidgenes associated with metabolism, a set of said oligomers, use of a setof oligomer probes comprising at least ten of said oligomers fordetecting the cytosine methylation state and/or single nucleotidepolymorphisms (SNPs) in a chemically pretreated genomic DNA, a methodfor manufacturing an array of different oligomers fixed to a carriermaterial for analyzing diseases associated with the methylation state ofthe CpG dinucleotides, a method for ascertaining genetic and/orepigenetic parameters for the diagnosis and/or therapy of existingdiseases or the predisposition to specific diseases by analyzingcytosine methylations, a kit comprising a bisulfite reagent as well asoligonucleotides and/or PNA-oligomers, and the use of said nucleic acidfor the diagnosis of metabolic disease, solid tumours and cancers (Oleket al., 2001).

[0011] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of PAZ/PIWI domain-containing protein.

[0012] Consequently, there remains a long felt need for agents capableof effectively inhibiting PAZ/PIWI domain-containing protein function.

[0013] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of PAZ/PIWI domain-containingprotein expression.

[0014] The present invention provides compositions and methods formodulating PAZ/PIWI domain-containing protein expression.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding PAZ/PIWI domain-containing protein, and which modulate theexpression of PAZ/PIWI domain-containing protein. Pharmaceutical andother compositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofPAZ/PIWI domain-containing protein in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofPAZ/PIWI domain-containing protein by administering a therapeutically orprophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding PAZ/PIWI domain-containing protein,ultimately modulating the amount of PAZ/PIWI domain-containing proteinproduced. This is accomplished by providing antisense compounds whichspecifically hybridize with one or more nucleic acids encoding PAZ/PIWIdomain-containing protein. As used herein, the terms “target nucleicacid” and “nucleic acid encoding PAZ/PIWI domain-containing protein”encompass DNA encoding PAZ/PIWI domain-containing protein, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of PAZ/PIWI domain-containing protein. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

[0017] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding PAZ/PIWI domain-containing protein. The targetingprocess also includes determination of a site or sites within this genefor the antisense interaction to occur such that the desired effect,e.g., detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding PAZ/PIWI domain-containing protein, regardless ofthe sequence(s) of such codons.

[0018] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0019] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0020] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0021] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0022] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0023] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0024] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0025] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

[0026] An antisense compound is specifically hybridizable when bindingof the compound to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. It is preferred that the antisense compoundsof the present invention comprise at least 80% sequence complementarityto a target region within the target nucleic acid, moreover that theycomprise 90% sequence complementarity and even more comprise 95%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarity. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0027] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare hereinbelow identified as preferred embodiments of the invention.The sites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

[0028] While the specific sequences of particular preferred targetregions are set forth below, one of skill in the art will recognize thatthese serve to illustrate and describe particular embodiments within thescope of the present invention. Additional preferred target regions maybe identified by one having ordinary skill.

[0029] Target regions 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

[0030] Exemplary good preferred target regions include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

[0031] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0032] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0033] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0034] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0035] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0036] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0037] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides from about 8 to about 50 nucleobases, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

[0038] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0039] Exemplary preferred antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

[0040] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare herein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

[0041] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0042] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0043] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0044] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0045] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0046] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0047] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0048] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0049] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF3, OCF₃, SOCH₃, SO₂CH₃. ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0050] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0051] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0052] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0053] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0054] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0055] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0056] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0057] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0058] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0059] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0060] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0061] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0062] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0063] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0064] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0065] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of PAZ/PIWI domain-containing protein is treated byadministering antisense compounds in accordance with this invention. Thecompounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0066] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding PAZ/PIWI domain-containing protein, enabling sandwich and otherassays to easily be constructed to exploit this fact. Hybridization ofthe antisense oligonucleotides of the invention with a nucleic acidencoding PAZ/PIWI domain-containing protein can be detected by meansknown in the art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of PAZ/PIWI domain-containing protein in a sample may also beprepared.

[0067] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0068] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0069] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

[0070] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0071] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0072] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0073] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0074] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0075] Emulsions

[0076] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

[0077] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0078] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0079] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0080] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0081] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0082] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0083] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of ease of formulation, as well asefficacy from an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive-preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0084] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0085] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0086] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtriglycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0087] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0088] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0089] Liposomes

[0090] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0091] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0092] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0093] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0094] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranesand as the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0095] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0096] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0097] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0098] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0099] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0100] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0101] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0102] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0103] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0104] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

[0105] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0106] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0107] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0108] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0109] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0110] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0111] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0112] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0113] Penetration Enhancers

[0114] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0115] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0116] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0117] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0118] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0119] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0120] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0121] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0122] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0123] Carriers

[0124] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0125] Excipients

[0126] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0127] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0128] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0129] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0130] Other Components

[0131] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0132] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0133] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0134] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0135] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0136] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0137] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy Amidites

[0138] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0139] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0140] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.) or prepared as follows:

[0141] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for5-methyl dC Amidite

[0142] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 μL) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol,1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05,0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂were added with stirring (pH of the aqueous layer 7.5). An additional 18L of water was added, the mixture was stirred, the phases wereseparated, and the organic layer was transferred to a second 50 Lvessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L).The combined organic layer was washed with water (10 L) and thenconcentrated in a rotary evaporator to approx. 3.6 kg total weight. Thiswas redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed bywater (6 L) and hexanes (13 L). The mixture was vigorously stirred andseeded to give a fine white suspended solid starting at the interface.After stirring for 1 h, the suspension was removed by suction through a½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cmCoors Buchner funnel, washed with water (2×3 L) and a mixture ofhexanes-CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h)to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.).TLC indicated a trace contamination of the bis DMT product. NMRspectroscopy also indicated that 1-2 mole percent pyridine and about 5mole percent of hexanes was still present.

[0143] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineIntermediate for 5-methyl-dC Amidite

[0144] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f)0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0145] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0146] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0147] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine PenultimateIntermediate for 5-methyl dC Amidite

[0148] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0149] THe product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was reequilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA (15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,coevaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.)to a constant weight of 2023 g (85%) of white foam and 20 g of slightlycontaminated product from the third run. HPLC indicated a purity of99.8% with the balance as the diBenzoyl product.

[0150][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC Amidite)

[0151]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 μg anoff-white foam solid (96%).

[0152] 2′-Fluoro Amidites

[0153] 2′-Fluorodeoxyadenosine Amidites

[0154] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0155] 2′-Fluorodeoxyguanosine

[0156] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0157] 2′-Fluorouridine

[0158] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0159] 2′-Fluorodeoxycytidine

[0160] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0161] 2′-O-(2-Methoxyethyl) Modified Amidites

[0162] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0163] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0164] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% dimer (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0165] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0166] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0167] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinePenultimate Intermediate

[0168] In a 50 L glass-lined steel reactor,2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and dimer DMT.

[0169] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA (25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0170] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T Amidite)

[0171]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0172] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0173] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0174] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0.51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0175] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidinePenultimate Intermediate:

[0176] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA (70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0177] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C Amidite)

[0178]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0179] Preparation of [5′-O— (4,4′-Dimethoxytriphenylmethyl)-2′-O—(2-methoxyethyl)—N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A Amdite)

[0180]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0181] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G Amidite)

[0182]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0183] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0184] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0185] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0186] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0187] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0188]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0189] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O2-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate(0.074 g, 0.003 eq) were added with manual stirring. The reactor wassealed and heated in an oil bath until an internal temperature of 160°C. was reached and then maintained for 16 h (pressure <100 psig). Thereaction vessel was cooled to ambient temperature and opened. TLC(EtOAc, R_(f) 0.67 for desired product and R_(f) 0.82 for ara-T sideproduct) indicated about 70% conversion to the product. The solution wasconcentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath(40-100° C.) with the more extreme conditions used to remove theethylene glycol. (Alternatively, once the THF has evaporated thesolution can be diluted with water and the product extracted intoEtOAc). The residue was purified by column chromatography (2 kg silicagel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, evaporated and dried to afford 84 g of a white crisp foam(50%), contaminated starting material (17.4 g, 12% recovery) and purereusable starting material (20 g, 13% recovery). TLC and NMRspectroscopy were consistent with 99% pure product.

[0190]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0191]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphen-ylsilyl-5-methyluridineas white foam (21.819 g, 86%) upon rotary evaporation.

[0192]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0193]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotaryevaporation.

[0194] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,NDimethylaminooxyethyl]-5-methyluridine

[0195]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0196] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0197] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

[0198] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0199] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0200]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0201] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0202] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0203] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0204]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0205] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0206] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0207] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0208] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0209] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0210] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl Uridine

[0211] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0212]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methylUridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0213] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0214] Oligonucleotide Synthesis

[0215] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0216] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0217] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0218] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. No. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0219] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or 5,366,878, herein incorporated by reference.

[0220] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0221] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0222] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0223] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0224] Oligonucleoside Synthesis

[0225] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0226] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0227] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0228] PNA Synthesis

[0229] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0230] Synthesis of Chimeric Oligonucleotides

[0231] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0232] [2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

[0233] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′--O-phosphoramidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by incorporatingcoupling steps with increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0234] [2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)]Chimeric Phosphorothioate Oligonucleotides

[0235] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0236] [2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0237] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0238] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0239] Oligonucleotide Isolation

[0240] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged-to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0241] Oligonucleotide Synthesis—96 Well Plate Format

[0242] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0243] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0244] Oligonucleotide Analysis—96-Well Plate Format

[0245] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0246] Cell Culture and Oligonucleotide Treatment

[0247] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0248] T-24 Cells:

[0249] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0250] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0251] A549 Cells:

[0252] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0253] NHDF Cells:

[0254] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0255] HEK Cells:

[0256] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0257] Treatment with Antisense Compounds:

[0258] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0259] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770)mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0260] Analysis of Oligonucleotide Inhibition of PAZ/PIWIDomain-Containing Protein Expression

[0261] Antisense modulation of PAZ/PIWI domain-containing proteinexpression can be assayed in a variety of ways known in the art. Forexample, PAZ/PIWI domain-containing protein mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0262] Protein levels of PAZ/PIWI domain-containing protein can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Antibodies directed toPAZ/PIWI domain-containing protein can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997).

[0263] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998). Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., (Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley-& Sons, Inc., 1991).

Example 11

[0264] Poly(A)+ mRNA Isolation

[0265] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0266] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0267] Total RNA Isolation

[0268] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0269] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0270] Real-Time Quantitative PCR Analysis of PAZ/PIWI Domain-ContainingProtein mRNA Levels

[0271] Quantitation of PAZ/PIWI domain-containing protein mRNA levelswas determined by real-time quantitative PCR using the ABI PRISM™ 7700Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0272] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0273] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each ofDATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverseprimer, 125 μM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM®Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-wellplates containing 30 μL total RNA solution. The RT reaction was carriedout by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0274] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0275] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0276] Probes and primers to human PAZ/PIWI domain-containing proteinwere designed to hybridize to a human PAZ/PIWI domain-containing proteinsequence, using published sequence information (GenBank accession numberNM_(—)024852.1, incorporated herein as SEQ ID NO:4). For human PAZ/PIWIdomain-containing protein the PCR primers were: forward primer:AAATTTGTCTCTCGGGTGAGTTG (SEQ ID NO: 5) reverse primer:TTAGTGCTGATTGGCTTGTCTAATTC (SEQ ID NO: 6) and the PCR probe was:FAM-AGTACTGACAGGACGGACTTTGCCT-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:8) reverseprimer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC— TAMRA 3′ (SEQ ID NO: 10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0277] Northern Blot Analysis of PAZ/PIWI Domain-Containing Protein mRNALevels

[0278] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0279] To detect human PAZ/PIWI domain-containing protein, a humanPAZ/PIWI domain-containing protein specific probe was prepared by PCRusing the forward primer AAATTTGTCTCTCGGGTGAGTTG (SEQ ID NO: 5) and thereverse primer TTAGTGCTGATTGGCTTGTCTAATTC (SEQ ID NO: 6). To normalizefor variations in loading and transfer efficiency membranes werestripped and probed for human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0280] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0281] Antisense Inhibition of Human PAZ/PIWI Domain-Containing ProteinExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

[0282] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanPAZ/PIWI domain-containing protein RNA, using published sequences(GenBank accession number NM_(—)024852.1, incorporated herein as SEQ IDNO: 4, GenBank accession number BF980145.1, the complement of which isincorporated herein as SEQ ID NO: 11, GenBank accession numberAI870324.1, the complement of which is incorporated herein as SEQ ID NO:12, residues 2461000-2587000 of GenBank accession number NT_(—)004568.7,the complement of which is incorporated herein as SEQ ID NO: 13, andGenBank accession number AK027796.1, incorporated herein as SEQ ID NO:14). The oligonucleotides are shown in Table 1. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target sequenceto which the oligonucleotide binds. All compounds in Table 1 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanPAZ/PIWI domain-containing protein mRNA levels by quantitative real-timePCR as described in other examples herein. Data are averages from twoexperiments in which A549 were treated with the antisenseoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”. TABLE 1 Inhibition of human PAZ/PIWIdomain-containing protein mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQID TARGET % SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO NO241284 5′UTR 4 52 agaacggagcccgccactgg 80 15 1 241285 Start 4 92ccgatttccattcatggagc 69 16 1 Codon 241286 Coding 4 196cttgaaaacagttagccagc 93 17 1 241287 Coding 4 230 tcatagaggtagacatcaat 5318 1 241288 Coding 4 285 gtcaaccacctccctgttca 83 19 1 241289 Coding 4290 attgagtcaaccacctccct 91 20 1 241290 Coding 4 307ctttaaaatgctgaaccatt 88 21 1 241291 Coding 4 344 ccatcataaactggtctacg 8822 1 241292 Coding 4 367 tggcggtgtaaagacttctt 84 23 1 241293 Coding 4374 agtggattggcggtgtaaag 77 24 1 241294 Coding 4 391ctgtagttgccacaggaagt 87 25 1 241295 Coding 4 403 ctaaatctacccctgtagtt 5226 1 241296 Coding 4 508 gtcctgtcagtacttcatgc 99 27 1 241297 Coding 4535 ctaattccagtggctcaggc 98 28 1 241298 Coding 4 574catcaacggcatggacaggg 86 29 1 241299 Coding 4 603 tttcatggagggcagatgtc 7830 1 241300 Coding 4 612 aggtgtgtatttcatggagg 75 31 1 241301 Coding 4641 tctggagcggagaaaaatga 51 32 1 241302 Coding 4 703gaacagactgatggaatcca 89 33 1 241303 Coding 4 911 ctcattgttccacaatgagt 5734 1 241304 Coding 4 946 gcctccttgttacattacaa 77 35 1 241305 Coding 41002 tctctccacagtttggccgt 67 36 1 241306 Coding 4 1037agagtatacttttctctgaa 68 37 1 241307 Coding 4 1052 gggtacttcagctgaagagt63 38 1 241308 Coding 4 1081 cctgcccgacttgcagacag 74 39 1 241309 Coding4 1214 tcttgtctatctggtgcaga 57 40 1 241310 Coding 4 1246aatttgcacttcttaccaat 64 41 1 241311 Coding 4 1258 gatctgtttcataatttgca53 42 1 241312 Coding 4 1277 tgaaactcctgaacaaatgg 61 43 1 241313 Coding4 1285 ctttaaattgaaactcctga 60 44 1 241314 Coding 4 1293atcccgaactttaaattgaa 59 45 1 241315 Coding 4 1300 ccatttcatcccgaacttta61 46 1 241316 Coding 4 1307 acatgagccatttcatcccg 76 47 1 241317 Coding4 1324 gaagtacgcgtccagttaca 74 48 1 241318 Coding 4 1363ctgtccgattccgtcctcca 52 49 1 241319 Coding 4 1388 catactccatggctcggtgt69 50 1 241320 Coding 4 1425 ttcaactcctgtgtggaatt 68 51 1 241321 Coding4 1496 gtgaaacccttcaatatttc 58 52 1 241322 Coding 4 1626gccagaatatgtgttcttga 69 53 1 241323 Coding 4 1644 gacgataataagctgtaggc59 54 1 241324 Coding 4 1741 ttacattcttgacttgaaca 69 55 1 241325 Coding4 1792 taacatttatctttaggcac 63 56 1 241326 Coding 4 1815aatattattgatccctccga 49 57 1 241327 Coding 4 1888 gtggatgagtgacatcggct54 58 1 241328 Coding 4 2031 aagttcccggaccatggagg 69 59 1 241329 Coding4 2104 cctctgaaacaccatcccga 57 60 1 241330 Coding 4 2136tagttcataatataatacct 59 61 1 241331 Coding 4 2169 ctccaaactgatgcaggctt12 62 1 241332 Coding 4 2202 tacaatgtaggttattccag 66 63 1 241333 Coding4 2237 gcacaaaataatcgagtgtg 66 64 1 241334 Coding 4 2259tccaaccctttctgtcctat 55 65 1 241335 Coding 4 2276 gggatattgccacttcttcc 066 1 241336 Coding 4 2310 gtgtgtaatgtctgtatcaa 65 67 1 241337 Coding 42512 ctaccaggtgagcataatac 56 68 1 241338 Coding 4 2617ccttggcaagagcttgtgga 62 69 1 241339 Coding 4 2650 ttgtgcgtaaggtatcttgg64 70 1 241340 Stop 4 2673 ttggactatttaagcgaagt 58 71 1 Codon 2413413′UTR 4 2697 agtacttcctctcagagaat 71 72 1 241342 3′UTR 4 2758ggaggtgtccttactcaatt 66 73 1 241343 3′UTR 4 2805 taaggatcagaccttggccc 5574 1 241344 3′UTR 4 2851 gatgctgtgttccttgatga 68 75 1 241345 3′UTR 42893 agaccgcacaaaaagcagtt 64 76 1 241346 3′UTR 4 2990caaagatagttgtgctgcca 71 77 1 241347 exon: 11 340 aggtctctttcatcgatatt 4178 1 exon junction 241348 exon 11 615 gctttcgttctaacaggagg 75 79 1241349 exon 11 645 tggtgaataaaatgacaatc 78 80 1 241350 intron 12 519cagaaatccaccacccaata 73 81 1 241351 intron 13 6210 ataccacgttctgatttccc80 82 1 241352 intron: 13 36470 aaccacctccctaaagaagg 79 83 1 exonjunction 241353 intron: 13 52136 aggtctctttctggaaaaca 89 84 1 exonjunction 241354 intron 13 57925 aagcatagaaactttcagtt 18 85 1 241355exon: 13 83268 gagactttacccgtcctcca 76 86 1 intron junction 241356intron 13 92802 accagtcagactctctgtgt 69 87 1 241357 intron 13 97081gaactcctgacctcaggtga 87 88 1 241358 exon: 13 113064 gtaggcttacctgtattcca37 89 1 intron junction 241359 5′UTR 14 72 cggcacaggactggagccgg 26 90 1241360 5′UTR 14 123 tgagcaaccgcactccgaaa 91 91 1 241361 5′UTR 14 129ttcccctgagcaaccgcact 77 92 1

[0283] As shown in Table 1, SEQ ID NOs 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59,60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 79,80, 81, 82, 83, 84, 86, 87, 88, 91 and 92 demonstrated at least 50%inhibition of human PAZ/PIWI domain-containing protein expression inthis assay and are therefore preferred. The target sites to which thesepreferred sequences are complementary are herein referred to as“preferred target regions” and are therefore preferred sites fortargeting by compounds of the present invention. These preferred targetregions are shown in Table 2. The sequences represent the reversecomplement of the preferred antisense compounds shown in Table 1.“Target site” indicates the first (5′-most) nucleotide number of thecorresponding target nucleic acid. Also shown in Table 2 is the speciesin which each of the preferred target regions was found. TABLE 2Sequence and position of preferred target regions identified in PAZ/PIWIdomain-containing protein. TARGET REV COMP SITE SEQ ID TARGET OF SEQ SEQID ID NO SITE SEQUENCE ID ACTIVE IN NO 157800 4 52 ccagtggcgggctccgttct15 H. sapiens 93 157801 4 92 gctccatgaatggaaatcgg 16 H. sapiens 94157802 4 196 gctggctaactgttttcaag 17 H. sapiens 95 157803 4 230attgatgtctacctctatga 18 H. sapiens 96 157804 4 285 tgaacagggaggtggttgac19 H. sapiens 97 157805 4 290 agggaggtggttgactcaat 20 H. sapiens 98157806 4 307 aatggttcagcattttaaag 21 H. sapiens 99 157807 4 344cgtagaccagtttatgatgg 22 H. sapiens 100 157808 4 367 aagaagtctttacaccgcca23 H. sapiens 101 157809 4 374 ctttacaccgccaatccact 24 H. sapiens 102157810 4 391 acttcctgtggcaactacag 25 H. sapiens 103 157811 4 403aactacaggggtagatttag 26 H. sapiens 104 157812 4 508 gcatgaagtactgacaggac27 H. sapiens 105 157813 4 535 gcctgagccactggaattag 28 H. sapiens 106157814 4 574 ccctgtccatgccgttgatg 29 H. sapiens 107 157815 4 603gacatctgccctccatgaaa 30 H. sapiens 108 157816 4 612 cctccatgaaatacacacct31 H. sapiens 109 157817 4 641 tcatttttctccgctccaga 32 H. sapiens 110157818 4 703 tggattccatcagtctgttc 33 H. sapiens 111 157819 4 911actcattgtggaacaatgag 34 H. sapiens 112 157820 4 946 ttgtaatgtaacaaggaggc35 H. sapiens 113 157821 4 1002 acggccaaactgtggagaga 36 H. sapiens 114157822 4 1037 ttcagagaaaagtatactct 37 H. sapiens 115 157823 4 1052actcttcagctgaagtaccc 38 H. sapiens 116 157824 4 1081ctgtctgcaagtcgggcagg 39 H. sapiens 117 157825 4 1214tctgcaccagatagacaaga 40 H. sapiens 118 157826 4 1246attggtaagaagtgcaaatt 41 H. sapiens 119 157827 4 1258tgcaaattatgaaacagatc 42 H. sapiens 120 157828 4 1277ccatttgttcaggagtttca 43 H. sapiens 121 157829 4 1285tcaggagtttcaatttaaag 44 H. sapiens 122 157830 4 1293ttcaatttaaagttcgggat 45 H. sapiens 123 157831 4 1300taaagttcgggatgaaatgg 46 H. sapiens 124 157832 4 1307cgggatgaaatggctcatgt 47 H. sapiens 125 157833 4 1324tgtaactggacgcgtacttc 48 H. sapiens 126 157834 4 1363tggaggacggaatcggacag 49 H. sapiens 127 157835 4 1388acaccgagccatggagtatg 50 H. sapiens 128 157836 4 1425aattccacacaggagttgaa 51 H. sapiens 129 157837 4 1496gaaatattgaagggtttcac 52 H. sapiens 130 157838 4 1626tcaagaacacatattctggc 53 H. sapiens 131 157839 4 1644gcctacagcttattatcgtc 54 H. sapiens 132 157840 4 1741tgttcaagtcaagaatgtaa 55 H. sapiens 133 157841 4 1792gtgcctaaagataaatgtta 56 H. sapiens 134 157843 4 1888agccgatgtcactcatccac 58 H. sapiens 135 157844 4 2031cctccatggtccgggaactt 59 H. sapiens 136 157845 4 2104tcgggatggtgtttcagagg 60 H. sapiens 137 157846 4 2136aggtattatattatgaacta 61 H. sapiens 138 157847 4 2169aagcctgcatcagtttggag 62 H. sapiens 139 157848 4 2202ctggaataacctacattgta 63 H. sapiens 140 157849 4 2237cacactcgattattttgtgc 64 H. sapiens 141 157850 4 2259ataggacagaaagggttgga 65 H. sapiens 142 157852 4 2310ttgatacagacattacacac 67 H. sapiens 143 157853 4 2512gtattatgctcacctggtag 68 H. sapiens 144 157854 4 2617tccacaagctcttgccaagg 69 H. sapiens 145 157855 4 2650ccaagataccttacgcacaa 70 H. sapiens 146 157856 4 2673acttcgcttaaatagtccaa 71 H. sapiens 147 157857 4 2697attctctgagaggaagtact 72 H. sapiens 148 157858 4 2758aattgagtaaggacacctcc 73 H. sapiens 149 157859 4 2805gggccaaggtctgatcctta 74 H. sapiens 150 157860 4 2851tcatcaaggaacacagcatc 75 H. sapiens 151 157861 4 2893aactgctttttgtgcggtct 76 H. sapiens 152 157862 4 2990tggcagcacaactatctttg 77 H. sapiens 153 157864 11 615cctcctgttagaacgaaagc 79 H. sapiens 154 157865 11 645gattgtcattttattcacca 80 H. sapiens 155 157866 12 519tattgggtggtggatttctg 81 H. sapiens 156 157867 13 6210gggaaatcagaacgtggtat 82 H. sapiens 157 157868 13 36470ccttctttagggaggtggtt 83 H. sapiens 158 157869 13 52136tgttttccagaaagagacct 84 H. sapiens 159 157871 13 83268tggaggacgggtaaagtctc 86 H. sapiens 160 157872 13 92802acacagagagtctgactggt 87 H. sapiens 161 157873 13 97081tcacctgaggtcaggagttc 88 H. sapiens 162 157876 14 123tttcggagtgcggttgctca 91 H. sapiens 163 157877 14 129agtgcggttgctcaggggaa 92 H. sapiens 164

[0284] As these “preferred target regions” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these sites andconsequently inhibit the expression of PAZ/PIWI domain-containingprotein.

[0285] In one embodiment, the “preferred target region” may be employedin screening candidate antisense compounds. “Candidate antisensecompounds” are those that inhibit the expression of a nucleic acidmolecule encoding PAZ/PIWI domain-containing protein and which compriseat least an 8-nucleobase portion which is complementary to a preferredtarget region. The method comprises the steps of contacting a preferredtarget region of a nucleic acid molecule encoding PAZ/PIWIdomain-containing protein with one or more candidate antisensecompounds, and selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingPAZ/PIWI domain-containing protein. Once it is shown that the candidateantisense compound or compounds are capable of inhibiting the expressionof a nucleic acid molecule encoding PAZ/PIWI domain-containing protein,the candidate antisense compound may be employed as an antisensecompound in accordance with the present invention.

[0286] According to the present invention, antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

Example 16

[0287] Western Blot Analysis of PAZ/PIWI Domain-Containing ProteinProtein Levels

[0288] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to PAZ/PIWIdomain-containing protein is used, with a radiolabeled or fluorescentlylabeled secondary antibody directed against the primary antibodyspecies. Bands are visualized using a PHOSPHORIMAGER™ (MolecularDynamics, Sunnyvale Calif.).

1 164 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 3050 DNA H.sapiens CDS (101)...(2683) 4 tgagtgcccg tcgcgtcgcg ccgcgtcgcc ccccgggccgcctccttgcc gccagtggcg 60 ggctccgttc tccctcgaag cactcccccc agctccatga atggaa atc ggc tcc 115 Met Glu Ile Gly Ser 1 5 gca gga ccc gct ggg gcc cagccc cta ctc atg gtg ccc aga aga cct 163 Ala Gly Pro Ala Gly Ala Gln ProLeu Leu Met Val Pro Arg Arg Pro 10 15 20 ggc tat ggc gcc atg ggc aaa cccatt aaa ctg ctg gct aac tgt ttt 211 Gly Tyr Gly Ala Met Gly Lys Pro IleLys Leu Leu Ala Asn Cys Phe 25 30 35 caa gtt gaa atc cca aag att gat gtctac ctc tat gag gta gat att 259 Gln Val Glu Ile Pro Lys Ile Asp Val TyrLeu Tyr Glu Val Asp Ile 40 45 50 aaa cca gac aag tgt cct agg aga gtg aacagg gag gtg gtt gac tca 307 Lys Pro Asp Lys Cys Pro Arg Arg Val Asn ArgGlu Val Val Asp Ser 55 60 65 atg gtt cag cat ttt aaa gta act ata ttt ggagac cgt aga cca gtt 355 Met Val Gln His Phe Lys Val Thr Ile Phe Gly AspArg Arg Pro Val 70 75 80 85 tat gat gga aaa aga agt ctt tac acc gcc aatcca ctt cct gtg gca 403 Tyr Asp Gly Lys Arg Ser Leu Tyr Thr Ala Asn ProLeu Pro Val Ala 90 95 100 act aca ggg gta gat tta gac gtt act tta cctggg gaa ggt gga aaa 451 Thr Thr Gly Val Asp Leu Asp Val Thr Leu Pro GlyGlu Gly Gly Lys 105 110 115 gat cga cct ttc aag gtg tca atc aaa ttt gtctct cgg gtg agt tgg 499 Asp Arg Pro Phe Lys Val Ser Ile Lys Phe Val SerArg Val Ser Trp 120 125 130 cac cta ctg cat gaa gta ctg aca gga cgg actttg cct gag cca ctg 547 His Leu Leu His Glu Val Leu Thr Gly Arg Thr LeuPro Glu Pro Leu 135 140 145 gaa tta gac aag cca atc agc act aac cct gtccat gcc gtt gat gtg 595 Glu Leu Asp Lys Pro Ile Ser Thr Asn Pro Val HisAla Val Asp Val 150 155 160 165 gtg cta cga cat ctg ccc tcc atg aaa tacaca cct gtg ggg cgt tca 643 Val Leu Arg His Leu Pro Ser Met Lys Tyr ThrPro Val Gly Arg Ser 170 175 180 ttt ttc tcc gct cca gaa gga tat gac caccct ctg gga ggg ggc agg 691 Phe Phe Ser Ala Pro Glu Gly Tyr Asp His ProLeu Gly Gly Gly Arg 185 190 195 gaa gtg tgg ttt gga ttc cat cag tct gttcgg cct gcc atg tgg aaa 739 Glu Val Trp Phe Gly Phe His Gln Ser Val ArgPro Ala Met Trp Lys 200 205 210 atg atg ctt aat atc gat gtt tct gcc actgcc ttc tac aaa gca caa 787 Met Met Leu Asn Ile Asp Val Ser Ala Thr AlaPhe Tyr Lys Ala Gln 215 220 225 cct gta att cag ttc atg tgt gaa gtt cttgat att cat aat att gat 835 Pro Val Ile Gln Phe Met Cys Glu Val Leu AspIle His Asn Ile Asp 230 235 240 245 gag caa cca aga cct ctg act gat tctcat cgg gta aaa ttc acc aaa 883 Glu Gln Pro Arg Pro Leu Thr Asp Ser HisArg Val Lys Phe Thr Lys 250 255 260 gag ata aaa ggt ttg aag gtt gaa gtgact cat tgt gga aca atg aga 931 Glu Ile Lys Gly Leu Lys Val Glu Val ThrHis Cys Gly Thr Met Arg 265 270 275 cgg aaa tac cgt gtt tgt aat gta acaagg agg cct gcc agt cat caa 979 Arg Lys Tyr Arg Val Cys Asn Val Thr ArgArg Pro Ala Ser His Gln 280 285 290 acc ttt cct tta cag tta gaa aac ggccaa act gtg gag aga aca gta 1027 Thr Phe Pro Leu Gln Leu Glu Asn Gly GlnThr Val Glu Arg Thr Val 295 300 305 gcg cag tat ttc aga gaa aag tat actctt cag ctg aag tac ccg cac 1075 Ala Gln Tyr Phe Arg Glu Lys Tyr Thr LeuGln Leu Lys Tyr Pro His 310 315 320 325 ctt ccc tgt ctg caa gtc ggg caggaa cag aaa cac acc tac ctg cca 1123 Leu Pro Cys Leu Gln Val Gly Gln GluGln Lys His Thr Tyr Leu Pro 330 335 340 cta gaa gtc tgt aat att gtg gcaggg caa cga tgt atc aag aag cta 1171 Leu Glu Val Cys Asn Ile Val Ala GlyGln Arg Cys Ile Lys Lys Leu 345 350 355 aca gac aat cag act tcc act atgatc aag gca aca gca aga tct gca 1219 Thr Asp Asn Gln Thr Ser Thr Met IleLys Ala Thr Ala Arg Ser Ala 360 365 370 cca gat aga caa gag gaa att agcaga ttg gta aga agt gca aat tat 1267 Pro Asp Arg Gln Glu Glu Ile Ser ArgLeu Val Arg Ser Ala Asn Tyr 375 380 385 gaa aca gat cca ttt gtt cag gagttt caa ttt aaa gtt cgg gat gaa 1315 Glu Thr Asp Pro Phe Val Gln Glu PheGln Phe Lys Val Arg Asp Glu 390 395 400 405 atg gct cat gta act gga cgcgta ctt cca gca cct atg ctc cag tat 1363 Met Ala His Val Thr Gly Arg ValLeu Pro Ala Pro Met Leu Gln Tyr 410 415 420 gga gga cgg aat cgg aca gtagca aca ccg agc cat gga gta tgg gac 1411 Gly Gly Arg Asn Arg Thr Val AlaThr Pro Ser His Gly Val Trp Asp 425 430 435 atg cga ggg aaa caa ttc cacaca gga gtt gaa atc aaa atg tgg gct 1459 Met Arg Gly Lys Gln Phe His ThrGly Val Glu Ile Lys Met Trp Ala 440 445 450 atc gct tgt ttt gcc aca cagagg cag tgc aga gaa gaa ata ttg aag 1507 Ile Ala Cys Phe Ala Thr Gln ArgGln Cys Arg Glu Glu Ile Leu Lys 455 460 465 ggt ttc aca gac cag ctg cgtaag att tct aag gat gca ggg atg ccc 1555 Gly Phe Thr Asp Gln Leu Arg LysIle Ser Lys Asp Ala Gly Met Pro 470 475 480 485 atc cag ggc cag cca tgcttc tgc aaa tat gca cag ggg gca gac agc 1603 Ile Gln Gly Gln Pro Cys PheCys Lys Tyr Ala Gln Gly Ala Asp Ser 490 495 500 gta gag ccc atg ttc cggcat ctc aag aac aca tat tct ggc cta cag 1651 Val Glu Pro Met Phe Arg HisLeu Lys Asn Thr Tyr Ser Gly Leu Gln 505 510 515 ctt att atc gtc atc ctgccg ggg aag aca cca gtg tat gcg gaa gtg 1699 Leu Ile Ile Val Ile Leu ProGly Lys Thr Pro Val Tyr Ala Glu Val 520 525 530 aaa cgt gta gga gac acactt ttg ggt atg gct aca caa tgt gtt caa 1747 Lys Arg Val Gly Asp Thr LeuLeu Gly Met Ala Thr Gln Cys Val Gln 535 540 545 gtc aag aat gta ata aaaaca tct cct caa act ctg tca aac ttg tgc 1795 Val Lys Asn Val Ile Lys ThrSer Pro Gln Thr Leu Ser Asn Leu Cys 550 555 560 565 cta aag ata aat gttaaa ctc gga ggg atc aat aat att ctt gta cct 1843 Leu Lys Ile Asn Val LysLeu Gly Gly Ile Asn Asn Ile Leu Val Pro 570 575 580 cat caa aga cct tctgtg ttc cag caa cca gtg atc ttt ttg gga gcc 1891 His Gln Arg Pro Ser ValPhe Gln Gln Pro Val Ile Phe Leu Gly Ala 585 590 595 gat gtc act cat ccacct gct ggt gat gga aag aag cct tct att gct 1939 Asp Val Thr His Pro ProAla Gly Asp Gly Lys Lys Pro Ser Ile Ala 600 605 610 gct gtt gta ggt agtatg gat gca cac cca agc aga tac tgt gcc aca 1987 Ala Val Val Gly Ser MetAsp Ala His Pro Ser Arg Tyr Cys Ala Thr 615 620 625 gta aga gtt cag agaccc cga cag gag atc atc cag gac ttg gcc tcc 2035 Val Arg Val Gln Arg ProArg Gln Glu Ile Ile Gln Asp Leu Ala Ser 630 635 640 645 atg gtc cgg gaactt ctt att caa ttt tat aag tca act cgg ttc aag 2083 Met Val Arg Glu LeuLeu Ile Gln Phe Tyr Lys Ser Thr Arg Phe Lys 650 655 660 cct act cgt atcatc ttt tat cgg gat ggt gtt tca gag ggg cag ttt 2131 Pro Thr Arg Ile IlePhe Tyr Arg Asp Gly Val Ser Glu Gly Gln Phe 665 670 675 agg cag gta ttatat tat gaa cta cta gca att cga gaa gcc tgc atc 2179 Arg Gln Val Leu TyrTyr Glu Leu Leu Ala Ile Arg Glu Ala Cys Ile 680 685 690 agt ttg gag aaagac tat caa cct gga ata acc tac att gta gtt cag 2227 Ser Leu Glu Lys AspTyr Gln Pro Gly Ile Thr Tyr Ile Val Val Gln 695 700 705 aag aga cat cacact cga tta ttt tgt gct gat agg aca gaa agg gtt 2275 Lys Arg His His ThrArg Leu Phe Cys Ala Asp Arg Thr Glu Arg Val 710 715 720 725 gga aga agtggc aat atc cca gct gga aca aca gtt gat aca gac att 2323 Gly Arg Ser GlyAsn Ile Pro Ala Gly Thr Thr Val Asp Thr Asp Ile 730 735 740 aca cac ccatat gag ttc gat ttt tac ctc tgt agc cat gct gga ata 2371 Thr His Pro TyrGlu Phe Asp Phe Tyr Leu Cys Ser His Ala Gly Ile 745 750 755 cag ggt accagt cgt cct tca cac tat cat gtt tta tgg gat gat aac 2419 Gln Gly Thr SerArg Pro Ser His Tyr His Val Leu Trp Asp Asp Asn 760 765 770 tgc ttt actgca gat gaa ctt cag ctg cta act tac cag ctc tgc cac 2467 Cys Phe Thr AlaAsp Glu Leu Gln Leu Leu Thr Tyr Gln Leu Cys His 775 780 785 act tac gtacgc tgt aca cga tct gtt tct ata cct gca cca gcg tat 2515 Thr Tyr Val ArgCys Thr Arg Ser Val Ser Ile Pro Ala Pro Ala Tyr 790 795 800 805 tat gctcac ctg gta gca ttt aga gcc aga tat cat ctt gtg gac aaa 2563 Tyr Ala HisLeu Val Ala Phe Arg Ala Arg Tyr His Leu Val Asp Lys 810 815 820 gaa catgac agt gct gaa gga agt cac gtt tca gga caa agc aat ggg 2611 Glu His AspSer Ala Glu Gly Ser His Val Ser Gly Gln Ser Asn Gly 825 830 835 cga gatcca caa gct ctt gcc aag gct gta cag att cac caa gat acc 2659 Arg Asp ProGln Ala Leu Ala Lys Ala Val Gln Ile His Gln Asp Thr 840 845 850 tta cgcaca atg tac ttc gct taa atagtccaag tatattctct gagaggaagt 2713 Leu ArgThr Met Tyr Phe Ala 855 860 actgaaagat gaattgacat acaacgtatg tttccagtgaagtcaattga gtaaggacac 2773 ctccagccat acagaaacca acactgtgtg ggggccaaggtctgatcctt atgttaacac 2833 aaggaagatt gtttacttca tcaaggaaca cagcatcattatgcaatatg aaaccagcca 2893 actgcttttt gtgcggtctc ctataggaag tatcgcaattgtcttgtttt catttcttgt 2953 agtctaaccc ttttaatgcc tttacctcaa gttgcttggcagcacaacta tctttgcaaa 3013 aaaaagtaaa gaaaaagtaa atgatggttt aaaaaat 30505 23 DNA Artificial Sequence PCR Primer 5 aaatttgtct ctcgggtgag ttg 23 626 DNA Artificial Sequence PCR Primer 6 ttagtgctga ttggcttgtc taattc 267 25 DNA Artificial Sequence PCR Probe 7 agtactgaca ggacggactt tgcct 258 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNAArtificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 676 DNA H.sapiens 11 gggtagattt agacgttact ttacctgggg aaggtggaaa agatcgacctgtcaaggtgt 60 caatcaaatt tgtctctcgg gtgagttggc acctactgca tgaagtactgacaggacgga 120 ccttgcctga gccactggaa ttagacaagc caatcagcac taaccctgtccatgccgttg 180 atgtggtgct acgacatctg ccctccatga aatacacacc tgtggggcgttcatttttct 240 ccagctccag aaggatatga ccaccctctg ggagggggca gggaagtgtggtttggattc 300 catcagtctg ttcggcctgc catgtggaaa atgatgctta atatcgatgaaagagacctc 360 tggcagcagt gtggagaata gatagaggag aaaaaactaa tctgagaagccagttaggag 420 gcttttcaat cactagttca ggtttcagtc tacatgttac ttccttgagaagcagtgttt 480 gacacccttc tcccccaatc cagccatccc ccaagtctga gttaggtatttctcttctgt 540 attcccatag cacagtgtaa ttcccctata atagcatgta tcaccttgaattatgggtgt 600 ttattgttct gtctcctcct gttagaacga aagctccatg aagggattgtcattttattc 660 accagtgtac cctcta 676 12 574 DNA H. sapiens unsure 356unknown 12 gacaagtgtc ctaggagagt gaacaggaag gtggttgact caatggttcagcattttaaa 60 gtaactatat ttggagaccg tagaccagtt tatgatggaa aaagaagtctttacaccgcc 120 aatccacttc ctgtggcaac tacaggggta gatttagacg ttactttacctggggaaggt 180 ggaaaagatc gacctttcaa ggtgtcaatc aaatttgtct ctcgggtgagttggcaccta 240 ctgcatgaag tactgacagg acggaccttg cctgagccac tggaattagacaagccaatc 300 agcactaacc ctgtccatgc cgttgatgtg gtgctacgac atctgccctccatganatac 360 acacctgtgg ggcgttcatt tttctccgct ccagaaggat atgaccaccctctgggaggg 420 ggcagggaag tgtggtttgg attccatcag tctgttcggc ctgccatgtggaaaatgatg 480 cttaatatcg atggtaaggg aactaaagcc atattctgta ttgggtggtggatttctgta 540 tgatgtgtgt acataaattt tatatataat tata 574 13 126001 DNAH. sapiens 13 aggagtttgg gttttcaagt gtgacgaggc gctattaaag agttttaaacatggaaatta 60 ctcgatctga tgtttgtgta gtgtcctgtt tgttagtcat cttttccaaactataataca 120 tttttttttt cctaaaaatg gtcgttaaag cagcctagga cttaggtcaattgcggtggt 180 cacttgaccg cctgtgagga ggcggcgcgt ggggtggatc ggactgggcgtggcgggggc 240 tcaggaagga gggtggccct accccagcgg gctcggctcg gggcctccgcggcagttcgg 300 ggtccttcac ccgccggctc caggtaggct actcctcagg taagccccgccgccagccgc 360 gacgtcgtcg cagacaggca ccgcccccac tcgtgcggcg cgagtagtcctgcccctccg 420 cgttgtctcc ggccggcacg gcccggcggg gtacggccga gcccgccgcatgtggcccgg 480 ctcccggaca cctccccggc gtcctccgcg ccggccgctc ctgccccgacgtcgctccgg 540 cacggctcgg ggcccagagg cgaggcgagg acgccgggca agccaggcagcggaactgac 600 gccggcgagc ttccggggcg gccccgggca ggtcggcggc ggcggcccgcagtcgtggag 660 gagcggtggg agcgtcggcg gccgcgggcg atgcaacttc cggacgggactcccctctgt 720 ccgcgcctca catctcccct tcctctcgcc tagtcctgtg ccgttttccgtccgcgactc 780 ttccggccca gagctttcgg agtgcggttg ctcaggggaa gccgtcgccgcccccgcctc 840 ggggccgagt gagagtgccc gtcgcgtcgc gccgcgtcgc cccccgggccgcctccttgc 900 cgccagtggc gggctccgtt ctccctcgaa gcactccccc cagctccatgaatggaaatc 960 ggctccgcag gtgagtcaga gtagctgggc caggtagggg atgtcacccagctactgtcc 1020 tctgagcatc cctgctcctc ccgcccggcc caggtgcgcg aggtgagcgtcgggcgggca 1080 tcgctcggtc tcccgcccct cgccctgctc ctccgcgacc tccccgcagcccagccccag 1140 ttccccgggg gcccctgagt cggcgaaact gcgaggcggg gaaacgcttggaggatttaa 1200 gtttggggtt atctaggcgg cattactctt tgctggagta cccttcttctagactttaga 1260 atggtttgcc attgtctagt tggagtgcgt gtcctttagc caggttgtgtgttccgtaga 1320 ggctgggcag ccagccagct cccttaccta cctcttagga tagttgtgaagataggctga 1380 gataacggat aacttcagat taagttcgaa ggaggtgttg gtgcaacgttaaattcaaca 1440 tggcattgct cctaccctcg agttcttttc tgttgtttgt ggcagcatggtagatagttt 1500 ctgaggagct tggaaatgtc atagcagcct ggatccctgc ttatacgaggaggtggttct 1560 taaaattgct gacagtatat tttttttcat tctctattcc ttagagaagtagttgtcata 1620 ttcctggaaa tttggaattt aagaaaactg ctttatctct gggggcaagagcagcagttt 1680 tgcagtctta agagaaaatt gcaacatgga tagtacttgt ccttaaaaaaggaaaagtat 1740 tgtttatggt tctaaagtaa ttaattcagg gacagaaagg tgttgcataacggtttgcct 1800 aacaaatgat catgcttggc ttattaaatt tgaaagtatg cttcagacgatcacaagttc 1860 gtaaattaat tttcaaaata tttgcggggc tgtcttgtta ctaatggtgttacaagttcc 1920 tgaattccat atctcttttt gccaatttga tataagaaaa acttacgtaaaagaaaccag 1980 tgacataacg atagctaaga actttgttga atagattacg tgtcaaatggttgcaaaaaa 2040 gcactcaaac tggggagatg agatggggag agagttacaa atttttttttttttttttta 2100 caaattttgt tacatggaag tttctctaat caatttagac catatctctttttcctgctc 2160 cattgtttcg ctttccccca tatcgtctta aaaggtctgt ctggattgtgaaaagatttc 2220 gactcttatt cagttttgat ttgtgtattt ctgaaactgt cctgcccttttaaaaaaatt 2280 accatactac tgttttactg tacaggagat gtgattattt gggaccataggtgatctttg 2340 tacataactg tcctgttgtc aagtctggaa aacaagtcat gaaggtcagaccctttatta 2400 atcatcccaa aacttttaaa gatatttcaa aaaagttttt aaagttttttctttttctcc 2460 ttagggtttt tcatatgata ttgtgcccat atatatggga aatgtcttagaaaacatttg 2520 ttacatcaaa ccacctagtc aactggtaca actaagccaa gtacagctcagaaaatacat 2580 tttacctctt cttttttggg tgtcccattg ctgaaatgga tacccatttagctgtcttca 2640 catttgggga agtgtgtagt tagattatca actatatgcc tgggtctcttgaaaagtaaa 2700 gttttttctt cagtacagtt tatacatgat tgaatgtagc ctaataatgaagataagcta 2760 tactttggct caagattgtc atcagaaaca aaattttcat tattcctgagacttgtatat 2820 aattggtatg cttagcttta agttgaaagc atagctgtgc aactaaattttaaatccata 2880 atttaggctg ggtgtggtgg ttcatgcctg taatctcagc actttgggaggctgaggaag 2940 gatgatcgct tgagcccagc agttggagac cagcctaggt aacatagtgagacctgcctc 3000 tacaaaaaac agaaaactta gccgagaatt gtggcacatg tctccagtcctagctacttg 3060 ggaggctgag gcaggagaat ggattgagcc cagaaggcag agattgcagtgagctatgat 3120 cgcgccacta actccagcct gggtgacgga gcaagaccct gtctcaaaaaaaaaaaaaaa 3180 aaaaaaaaaa aaaaaaatca tcatttgaca cgtattttga ttttaaaattttagtataaa 3240 tgttctcaaa aagtctattg actcagtatt gaatcttggc tttccaattctgccataagt 3300 ttttcttttt tgtggagatt ctcaggaatc atcttgtgat aggatattttaaattctagt 3360 tgctcggtgg atacacagaa aactgttaaa tttgtttaca ttagggaacctcgaacacaa 3420 ataccatata agttagtatt tgttctttca tatgaaatat tttaaatgctttttaaggat 3480 atcttgtgca atctactgat ttagtaccat cagaactttg atttaacaaaaaaaaaggtg 3540 aaattagtta aaaattaatc aacagtttaa catctgtgtc tgtgactctttagttgggga 3600 acatctgatg aaacaccgtt ttttatggtc taattactgc tgaactaagagtataggtta 3660 tgtttcagtt ctaaataaag tgggaatagg gagctgcaga aatgcttgatgatgttactt 3720 ctcctgagta ctcatttttt aggtccatac tattctagtc tatgttttcaggtaatgtta 3780 gactaccgtt taggacagag gaactacact ttataatgga agaaaaacatttacttttac 3840 cttaaattaa tgacatgcaa attgtttgat gtttggtgga agttttatacagtcttgcaa 3900 aagtgaatag actgtttctt tcttatttac acttttaaat tcatgtctgaaaatagttgt 3960 tttagagttc tgtgtttatt taataataaa agttttagaa aagttatttggaaccaagtt 4020 ccaaaggaat aaaagttgca tatatgggaa gctagaacta aatcaagattgggatttgaa 4080 tgatgtagat aaaaattatg ggtcaaaaat acctactaaa atatgtaatgccatcagggt 4140 cccaatttag tatttaaaac aatcttcttt ttatttattt attttttttgagatggagtc 4200 ttgcttgctc tgtcgcctag gctggagtgc agtggcatga tattggctcactgcagcctc 4260 tgcctcctgg gttcaagtga ttctcttgtc tcagcctccc gagtagatgggattacaggc 4320 gcgcaccacc atgcctggct aattttttgt atttttagta gagacggggtttcaccatgt 4380 tggccaggct ggtcttgaac tcctgacctc aggtgatctg cctgcctcggcctcccagaa 4440 aacaatcttc tttaataact ttgagaaatg tttttgtcgt tatataaattttctcacttg 4500 tagttctttg ccttgaagaa aaaaaatcaa attactgctc tgtctgtgggcatgaatttt 4560 gaaagtgata aaggtttttc tactgggttg tcacttaaat tttggcttatgctgccctgg 4620 agtgaccatt atgacttaaa aaaaatatat ttattgcact tgactggcagtgggacttat 4680 aaaaatgtgc aagattgttt tgatatttgg tttagaattt ctctttcaatgaagggactc 4740 cgagggaaaa caaaaattga gacataaaat aaatttttcc ttctaaaaaaagacttaagg 4800 gaaaagacat atagataacc ttcaaatata attcagttaa catgaagacatttacaaaat 4860 tacccatgat tctatcagcc tcatataacc gttctttaca tttttaaaccaaattctaaa 4920 ctaatctgtt tagtttttta aaacttaagg aattaagtta tttaggaaagccattagaat 4980 gaatgaaaag atatctacat gctacttcat tttgcttttt tggagaattgtatgtaataa 5040 gttggtaatt tagaattaat ttgtattagt ttatatcttc caatgggaacattgcgtttt 5100 ttagttactg cctggcctga cagtatgcaa agaggcctgg ggccctgtaaaggggacatt 5160 tcctggaaga attctctgtt gtatatggaa aaaccctggc tcagagtagatttcttgcca 5220 aattgtctaa gctgatggat tctagttgaa taccattttg ctttataatatagccatcta 5280 gtttcacatc ggtttctata ttcttaaaat acttgaggat ctacctgaaaggtgaattaa 5340 aatattatta atctaacaaa acatattatc ttatagtact taaaactagaatcttaaaaa 5400 gtaatttata agtttttgtt gttgctaatg ggtaggggaa aaaagaccaaagtgataatt 5460 ctgttttcag gaaggtaaaa ctaaaaagat aatttattta agatcagtatcctttccaga 5520 cctgtttagt ctcagactgt tgagatgaac agcatttacc agacattcctaccttctaat 5580 tcagttgtct tggatactga atagaccctc attcttggct cattaacaaaacagatgtga 5640 gaaagaatat tgtgtggttt ttcagccata atggttatgc tgttaggatacatgaacagc 5700 ctttctgtat ttggagtttc tgtggttttc tgccattatc tgtgttaatattaatgactt 5760 tctttggcta gccactgctt aaaaaaaaaa aaccaactat tgagattcagcaaaaccttg 5820 tcacacaact gatgctttct cttatacatt aaaatgtggg cattttgtggtttggttata 5880 ataaaaaaaa gtgtgcatta gtctgaaatg tcagtttaag gaaatgaagaattccttgtt 5940 ttttgtttgt ttgtttgttt gagacagagt tttgctctcg tcgcccaggctagagtgcaa 6000 tggtgtgatc ttggctcagt atttttagta gagatgggat ttcaccatgttggccaggct 6060 ggtcttgaac tcctgacctc aggtagtcca ctcacctcgg cctcccagagtgctgggatt 6120 acaggcatga gccactgcac ctgccctaga attccttgtt ttacatcagccagttatttt 6180 atacatcatt tccaaattgt caagttcttg ggaaatcaga acgtggtatctacagtctat 6240 tgatgtgaga catttttaga ttaaaaaaat attttttgta gagacgggatcttactatgt 6300 tgcacaggct ggtcttgaat tcctaggctc aaatgatcct cctgcctcagcctcccaaaa 6360 tgctgggatt gcaggagtga gccaccatgc ccacccatat ttttaggtttttcatttgta 6420 gaagaaattt tacaagaatg tgttctcaat tgtaagctta cataatactacttttgagtc 6480 attactaata cttggtattt taactgattt ctgaatcttc taacaatatgagagagacat 6540 agtatttctg tgaactttaa aaatgatgaa agaatagatt gcaaaatgggctcttactaa 6600 taacaaggga aatgtcccct tttattttca agggaggaaa tgccttttaaaaattgtttc 6660 tcactcctgt aatctcagtg ctttgggagg ccgaggcagg tggatcagctgaggtcagga 6720 gttcgagact agcctgacca aaatggtgaa acctcgtctc tactaaaaatacaaaaaatt 6780 agccgggcat agtggcgggt gcctgtaata cccgctactc gggaggctgaggcaggagaa 6840 tcctttgaac atgggaggtg gacattgcag tgagttgagg tcacggcattgcactccagc 6900 ctgggcaact caaaaaaaaa aaaaaattgt ttcccagccg gatgtggtggctcccacctg 6960 taatcccagc actttaggag gctgaagcag gcagatcatg aggttaggagttcaagacca 7020 gcctaaccaa catggtgaaa ccccatctct attaaaaata caaaaattagctgggcatgg 7080 tggcgtacac ctgtaattca gccacttggg aagctgagac aagagaattgcttgaacctg 7140 ggaggcggag gttgcagtga gccaagatcg tgccactgcc ctccagcctggaccacagtg 7200 cgagattctg ctcaaaaaaa gaaataaatt gtttcccata ctgccacctgataagcttaa 7260 ccctcaactg gctggatgtt ctataagtga ttatttaatt gtaatgagcctaataataag 7320 tgcggtatgt ttggacagat tcattgaatg aaaaagtgga attagcaggtaggaggttcc 7380 tgaagttcca tgctgtttac tacgtagctt tgcagactta acatgtataaaatcagagac 7440 atttcattaa gtcagatttt gagatcaaca caatatattt ctttttccaaaacaaaaatg 7500 tattcttttt tttttttttt tttgagacgg agtcttgctc tgtcgcccaggctggagtgc 7560 aatggcatga tctcggctca ctgcaacctc cgcctcctgg gctcaagcaattatcctgcc 7620 tcagtctccc tagtagctgg gattacaggt gcccgccacc acgtccagctaatttttgta 7680 tttttagtgg agatggggtt tcaccatgtt agtcaggctg gtcttgaaccagacctgacc 7740 tcaggtgatc cacccttctt ggcctactga agtgctggga ttacaggtatgagccactgt 7800 gcccggccca aaaatgcatt ctttttccaa ttataaaata ataactacatgtttattact 7860 ttaaaaaaca aacgatataa gaatgtctca aatagaagat gaaagtatgatcctatcctc 7920 cagatgaaac cattgttaac tctttcttgt atatcttccc agacatccatccgtctgtcc 7980 atatatttat catacgaatt gtttctaacc ttctttttcc acttagtaatgtgtcgtaag 8040 tatctttccc atatcattac ttacatctat ataatagtat aataatttatactgagtaca 8100 tagcatttaa ttttatctgt atattgatca gtctcattga tagtggtttagattttttcc 8160 agttttttgt tattatgact aaaactttgt aagtattcta gtacatatgtgtttgtatac 8220 tggtccagtg cttgcttttg gataaattgt tagaagtaga attattgaaacagtattcca 8280 tgaatattaa agaaaatgtt tccagtgaaa atctataagt tagtaattggctatagtata 8340 tgttatagtt gattttgatt tattcactgc ttgttttttt tcatcagtcacatttgctgt 8400 aggctattgt ttagctttag actttccaac tggtacacat tggattactagatgagtgaa 8460 caacatggac acatgtatgc tttggaaatg tatggtttta tgtttgaaatttagtttggt 8520 tagttattat ccagtacata caataactgc tgaaagaaaa gtttgatatagggagaaagt 8580 ccagatagtg ctttgtattt ctgtgtagtt atatttccaa ctctagtgggcagtatgtat 8640 ttgttaaata actaaaatat gcttcattgg aagtataatt cattgtattgacagaattgt 8700 ttcatctgct aatttacatt attatgtaat gtaaatattt cataatattctggatattat 8760 gaaaatatcc agaatatttt ctggatacta aacttgatta gtatctatagaattctgttc 8820 attgcttatt catgcaacag aattttgctt tgtgccaaat tatttaaaaagcaccaggta 8880 aagtaatgac catggagaaa aaaattgaca gtatgatata gtgtaaaaaacatgggtttt 8940 agagacagat tctggctctt aaattaactg aaatttatta atgatgtgtcggtataggtt 9000 tgagtgcaaa tgttctcctc ttgtagagga tgttgatagt agggtgtctgtgtgtatgtc 9060 agggcaggag gcctgggaca tatgggaaat ctctaccttc tgttcaattttgctgtgaac 9120 ctaaaacttc cctaaaatag tctatcagaa aaagttaact gctactttgggcagtgcatt 9180 taatcttcct taaccttaat tttcttatct gtacaatggg atagtaagaagagatgacac 9240 atgcaaagga aatggccatt tctctctttt ttatgatatt ttactatagagaatttagga 9300 tgtatacata taggcagaac tgtataataa actctattgt acccaccacccaaacgcagt 9360 catcaaccca cgtccaatcg cttctcttct acttttccct cttttatatttttgaagcat 9420 attctaggta taatatgatt ttattcatat ttagtagtaa ctataaaagttatggactca 9480 tgatatagta ccattatcac agcaaaataa taatcactta taaaaatttctaatcattgt 9540 tcaaattttt acttgtctca catattattt tttaaactgt ttgccttaaaaaaaaatttt 9600 tttttttttg agatacagtc tcactctgtt gcccaggctg gagttcagtggcatgatctt 9660 ggctgactgc aaccttcacc tcccaggttc aagcgattct cctgcctcagcctcctgagt 9720 agctgggatt acaggcatgc gccatgacgc ccggctaatt tttgtatttttagtagagac 9780 ggggtttcac catgttggcc aggctggtct tgaactcctg acctcaggtgatccgcccgc 9840 gtcagcctcc cagagtgctg ggattacagg catgagccac cacgcccagcctaaaaaaaa 9900 tttttgattc aaaatccaaa taagttctac acattgtgat tgatcgatatgtcttttaag 9960 tgtcttaatc ggtaagtttt ctctccttgt ttttctcctc tgcaatttatgggctgtttg 10020 tccttttaga atttttcacg atctggattt tgctggttcc atctttacaatttaatttaa 10080 cataatcctt tgaatttcct gtaaactgct agtggatcca agggcttgatcaaattcagg 10140 ccattctttt tgaacttact acagttgggt ttttgtccct agcacttgactggaattgtt 10200 tttatcaagg tcagcaaaga cttagctaaa cccaatagtt cccagatcttcattttattt 10260 catccacatc taatgacatt ttcttcttga aacactgttt ttctccatttggttttcagg 10320 gtaccactct ctccaggtcc tcctccaacc ttgttggctg ttacttttccagttcctttg 10380 ctgttttcat ttccctaatt tctaaatatt gggagtatcc ttggggttagtattctgtat 10440 ccatgccaca gtctgtctga tctctaatcc agtggtttta aataacacttctatgctgag 10500 gacacccaca tttacacctc cagcctggac ctctcctctg aactccacactcatctaact 10560 gcttactatt catctacttg tagacacctc aaatttagca tatccttaaaatcctcttga 10620 tttcccctcc aaacttgcta ctatcactga gtctttccta tctcagtaaatgacacttct 10680 gttctttcag ttgtacagac caaaaaacct tgagaatgtt tctcctcataccccacatct 10740 agtccattta acaattcctg tcaggcctac cttctaaatg ttttccacatccacatcact 10800 cccctaactg tactactgta gttctagcca acattatctt tcacctagacagccacatag 10860 tctgctgact ggtctccctg cttgtaccct tatgtataat ttttcatacagcagctggag 10920 tgatacttaa aaaaaaattt aagtcagatc ataacacttt ttcactcagaactcagaggg 10980 ctgacactat ctaactaact tcaagactca caataaagct actgtaatcaagataatgtg 11040 gtattggtga aagaaacaac taatggaaca gaacagagag tccagcatggcaggttacca 11100 tcccaattct cctacttact agctataaaa attttaggca aattatttcagttttcctca 11160 tctgtaaaat gattccttcc tttatagggt tggtatgaag attaaatgagataatgcatg 11220 taaaagcacc tagcaggcca ggcttggtgg ctcacacctg taatcccagtattttgggag 11280 gccaaggtgg gcagatcagt tgttctcagg agttcaggac gagcctgggcaacatggtga 11340 aaccctgtct ctaccagaaa tacaaaaact tagccctgca tggtgtaaaataaaagcact 11400 tagcacattg cctgagacat agtcagaact tgataaattt tagaatttgtggattttcta 11460 agttgatctt gacaagtttc ataagaaaga ggcagatcaa gtattattttcattttttag 11520 atcaggaaac aaattcaggg acagtatttg gtgacagtca aatgattagataattggcag 11580 agccagtact aagggctagt acggaatttg tacagtatta cttatctcaggctaggatag 11640 gaaagattat gccctctgaa gagattttta aaaaaacaca aagcggaatttaaaaacaaa 11700 tgattcaggc agcattttag tctcttttca tctacactga ataaaagttattgttagccc 11760 aattttttat tcctgataca aactcattct tttgatatat tgttggatttaatttaacat 11820 tttgttagga tttttgcatg tatattcgtg aattagatta gcttatgattttcctttttt 11880 atagtgtttt tgtcaggttt tagtaccaga atttttctgg catcataaattgtattgggg 11940 tgtgtttact tcttttctgt tctctggaag tgtttgtata acatcggtgttatttttttc 12000 cttaaatgtt tggtataatt ctctagtgaa gccatctgga tctggaatctttttgtatgt 12060 atgtggaagt attttaaatt gtggatttaa tttatttgga ggacttttaagattttttta 12120 atgtaatttt tagtttattc agataatagt ttattaattt attgtttgaattttgattaa 12180 caaagctgta ttttgagctt caaaaattta gtgctggaca tcacaggttttctttaaatt 12240 tttttgatta aaaaatgtaa aatatacaac cattaaattt accatcttaaccatttttgt 12300 gtatagtcag tagtgttcgg tacattcaca gtgttgtgca gccaatctgcacaactcttt 12360 tcattttcta taactgaaac tatatccatt taacaaatct gcatttgctcatatctcaac 12420 tcccggtgac cacacgtcta cttcctgttt cttatgaaat tgactactctaggtacctca 12480 tgtaagtgga atcatataaa ttatatagtg tttgtcattc tgtgactggttttgtttttt 12540 tttttgagac ggagtctcgc tctgtcaccc aggcgggagt gcagtggcatgatctcagct 12600 cactgcagcc tccacctccc aggttcaagt gattctcctg cctcagcctcccaagtagct 12660 ggtggctaca ggtgcacaca accacaccca ggtaattttt ttgtatttttttggtggaga 12720 tgatgtttca ccatgttggc caggctattc tcgaactcct gacctcaaatgatcctcccg 12780 ccttggcttc ccagagtgtt gggattacag gcgtgccatg cccatctttttttttttttt 12840 ttttgggaca gaatctcact tagctgccca ggctggagtg cagtggcacaatctcggctc 12900 actgcagcta ctgtctccca ggttcaagca gttctgtcat cccagcctcccaggtatctg 12960 ggattacagg tgcctgccat catgcctggc taatttttga attttagtagagacagggtt 13020 tcaccatgtt ggccaggctg gtcttgaact cccaacctca ggtgatccacctgccttggc 13080 ctcccaaagt gctaggatta caggtgtgag ccaccacccc cagccctttttttttttttt 13140 ttttaagtaa aagggtctcg cgctatcacc caggctggag tgcagtggcatgatctcggc 13200 tcactgtaac ctccacctcc ggggctcaag cgattctacc acctcagtcttctgagtagc 13260 tgggactaca ggtgcacacc tggctaattt tttgtgtttt tggtagatacaaacggggtt 13320 ttaccatgtt gcccaggctg gtcttaaact cctgagccca agcagtctgcccacctcggc 13380 ctcccaaagt gctgggatta caggtgtgag ccaccatgcc cggcctgtcttatttcactt 13440 aacataatat cttcaaagtt aatcatgttt tagtgtgtgt cagaatttcttttttaaggc 13500 tgaataatat ttcattgtat gtatatacca cattttgttt attcattcatctatcagtgg 13560 gtacttaggg tgtacaaata actcttcaat cagttctttc tgctttcacttcttttgagt 13620 gtatacccag aagtagaatt gctagatcac atggcaattc tgtttttcattttttgagga 13680 accatcatac tgtttttcaa agtgagtgta ccattttata gttccaccaacagaggactt 13740 ttcaaatttg atgaataata cttttttttt tttaagacag ggtcttgccattttgcccag 13800 gctagtctca aactcctaag ctcctaagcc tcctgagtgt ctgggattacaggcacaaga 13860 tgctgtgcct ggctatatag tactttagtg tatcagtaag ttttatttctctaggaattt 13920 gtctgtttca tcaaactgtc aaatttattg acacaatgta atgtcttcagtagctgtagt 13980 gacttttttt cacttctgtt attagttaag cccttttcct cctttttaaaagaaatcagt 14040 gttgcctaga ttttatcaat tttatttact tttttttttt tttttttttttggtaagaca 14100 gagcctcact ctgttgccca ggctgaagtg cagtggtgcc tcagcctgctaagtagctgg 14160 aactacaggc ctgcaccacc atgcctggct aatttttgta tttttttaaatagagacagg 14220 gtttcaccat gttggccagg ctggtcttga actcccaacc tcaggtgatctgcccgcttt 14280 ggcctcccaa agtcctggga ttgcagacgt gaggcactgc acccagcccacttatctttt 14340 tttgtttttt tgttacgggg tctcactctg tcacccagac tggagtgcataatcttggcg 14400 cattgcaaac tctgcctcac aggctcaagc cattctccca cctcagcctcctgtgtagct 14460 gggaccacag gtgtgcacca ccacactcag ctaatttttt gtatttttggtagagatggg 14520 gttttgcctt gttgcccatg gtggtctcaa actcctgagc tcaggcgatccacctgcttt 14580 ggcctcccag agtcccaaag tgctgggatt acaggtgtga gctgccatgcttggcctgag 14640 atacttacct tgtcaaataa tcaaataatt gattctgtac tttcttgattcttttttatt 14700 acatattgct tcttatttta ctcttttctt ttcttttctt ttttttttttaaatagagac 14760 aagttctctc tctggcactg agtctggagc gtcgtgttac tatcatagctcaagtgatcc 14820 tcctaccttg gcctcctaaa gcactaggat tccaggcatg agccaccatgccctgccctt 14880 tcttctactt tctttaactt tactttgcta ttggtctcac actctttaggtctgacttgt 14940 gcttgctcat ggtcagcttg gtttgtgtga ttgttcttcc ttctctctgccctttccaga 15000 acagttttcc agaggcatac ggttttctgt gtttattttt aatgaagagaggaaatctgg 15060 ggtgacagaa gttgaagtgt tagaaatgat gtcatcttgc agaactaggttttagccatt 15120 atcagctgta cttataattc taatatactc tggttgacaa ggctttggaatacagcttgt 15180 aactgtgact cttttttttt ccctttcccc tggcaggacc cgctggggcccagcccctac 15240 tcatggtgcc cagaagacct ggctatggca ccatgggcaa acccattaaactgctggcta 15300 actgttttca agttgaaatc ccaaagattg atgtctacct ctatgaggtagatattaaac 15360 cagacaagtg tcctaggaga gtgaacaggt aagaatcatg aaactgcaaagatcttttgc 15420 tatttttttc cttagtaatt atccatgttt attttgtata tctgaataacaattacaatg 15480 tgtaacagtt tgaccaaaaa catctggtaa tttgttttaa aactgattgtacttcagggg 15540 tgtgatagtg gggaaaaaat ctttgaaatt attttgttat aacacgagctcacattttcc 15600 ctgtgataat agaaaaggtt caagttattt ttacatgctc ctgaaatcaggctgcacatc 15660 atgagcacat cattttcctt gctgttaggt aatatgtcca tgcttgcttttttctcctca 15720 cctctcttat gtaccacttt cataatgttc cctttaagat gacggtggtgatgatagcag 15780 ttgggggtag aaatactggt ttcatgcttt cttttctcct ttcccaattcccaactgttt 15840 cttaccatta tataggaata agtacagatg gtatataaag atttatcagcctgctttcag 15900 taagcttcct ctcgcctccc ccaaatgcca tttatattct tggatgtggtttcggtaata 15960 caggaaatat aagaggaatt tatgattgga tatatactat gtctatttggattttgtttt 16020 taaaaacaaa gacaacacat tttaaaaaaa tgtgatattc agtttagcattttggtttct 16080 atgatcccag cctcttctta ttcattaaat gttattaaga gtcttcatttaagacattta 16140 agaaaaagaa tgttgtttct ctcaagaaat ttgtaatttg gtagaggaaataagacatgt 16200 aagaagtatg aaggtctttt tcttggactt gtcatcctaa ttgttagtatttctgttata 16260 cctgaaagtg aatgagcact aaaagacttt gtgataacac gttaaaaacaacaacacctc 16320 taagaatgtt gtaattaaca tgtaatgcag agttctttgt gaggtcaggaagactcgtag 16380 agtgttacag ttgaaaggaa cctcttaaga agttaactaa tccaataccttttttattta 16440 tagctgaaat ccagaaaggt taagggattt agtcaagaac acatgtttcaagtaggaaag 16500 gtgaaaccag aactccagaa tcctgacttg gtagtcatta ggaagtattttgtaaaggaa 16560 gaagttaatc taaaacaatt ggtaaaattt tggttagctg tagtaaaaataacatacata 16620 taattttatt tatttattta tttatttatt tttgaaggaa ggatagcagtaacaaaagca 16680 taacggttga gaagaaccag gtgtattgag tctgaaccag gcttaaggggaaggttcttt 16740 gcattacatg ttaattacaa cattcctata ggtggttttt tttttaacatgttatcagat 16800 actgctttaa gatactatct gttgaataga tgactcctaa tcttgctgtagcctctacct 16860 ctttcctaaa cttcagattg acacccaaat gttcctaaag tgaacaacacaagtcaaaag 16920 agtagtgccc tactcttctg tgacctcgta gtacttgatg tacacttccattgtggtgtt 16980 tttagtgcat tgtggttatt tttctccctt tctccagtat aaccccttcatgaaaagtag 17040 tgttttttat catctttgtc accccatgtc agctcagtga ttggcacataatactcagta 17100 aagtgaatga aatgtttata aaatggcatg agtgtgatgg tgggggacaggaaagaaaac 17160 tggatgttct agagggctgt tttcctgaac atgggttttt cagtgccgtactcttatctc 17220 ctaggatctt aggctagtct tggttttggt cttccttcac tctcttttatgtctttctgt 17280 gagccgtcat ccacttatgg ggacttaact gactcatgct gtactctcatagagattccc 17340 tttttcttgt ttctcaaaac tggttcatta atgtatagat ttgagtgaaggataatcctg 17400 acccttctgg tagatattta cactttaaaa aggcatttat tagctgagccatgggcctgt 17460 agtcccagct actctgaagg ctgaggcagg aggattactt aagcccaggagttaaggctg 17520 cagtgtgttg tgattgtgcc tgtgaacagc cactgcactc cagccggggcaacatagtga 17580 gaccacatat ctaaaaaata aataaataaa aataaaaagg cattagttaatctaagtaaa 17640 gatctggggc ttgagggatg tcaattcaag tttttttagt atgccagatgagttacaagg 17700 aaatgatatg gtggcaaaaa tattaagtaa aaggaacaga attttttttttttttttttt 17760 gcaacagagt ctctccctgt tgcccaggct ggagtgcaat ggcgcgatttccgctcactg 17820 caacctctgc ctcctaggtt caagcaattc tcctgcctca gcctcccgagtagttgggat 17880 tataggcacc tgccaccatg accagctaat tttttgtatt tttagtagagactgggtttc 17940 tccatgttgg cccaggctgg tcttgaactc ctgaccttgt ggtccccccccgcccccacc 18000 ccaccgccgg cctcccaaag tgctgggatt acaggcgtga gccactgtgcccggccggaa 18060 cagaattgtt tattagattt attctttctc ctgcctaaca tttttgttttattttttggt 18120 agggtgtggg taaaggaaaa tttgtgtatt atatatgtgt aattatatatgtgtatataa 18180 agttgtgtag catagaattt catgtgtata tgctcaatgt attaagtttgtgagaaaaat 18240 atgtgtcata aattggtgtg gtgcattctg gttttaactt acgattcttttgacatcctt 18300 aattacatta catctttagg ttgctttatc ttacattttt ttgacagtaccaataattta 18360 gatttgtaat ttaaatgttt ccaagtggaa agtttttaaa tttttgtgatgtaaaatttt 18420 agtcattttt atcatgtttt tgagttttta tttattttat tattattttatttatttatt 18480 tttttgagat ggagttttgc tcttgttgcc caggctggta tgcaatggcacgatctcggc 18540 tcattgcagc ctccatctcc tgggttcaag caattctcct gcctcagcctcccgagtagc 18600 tggaattaca ggcatgtgcc atcatgccca gctaattttg tattattagtagagatgagg 18660 tttctccatg ttggtcagtc tggtctcgaa ttcccaacct caggtgatctgcctgccccg 18720 gcctcccaaa gtgctgggat tacaggcgtg agctaccgca cccggcccaagtttttatag 18780 gaactgtagg acttgtttgg gattctaaaa atcatataaa tcagctttatactttgttaa 18840 taatattgct ttgatttaat gtcaacatct gcaaaactta ttccgttttcttcagctgac 18900 ttgcttttgt cttcagtttt tacagtatta ctgcatgact agtcagttgaaacttggtgg 18960 tcttctgaaa ttgatgtggt ggctcagttc ctgtgcttca acaactggaattctaggcgt 19020 tagaaggaac tagatagaac ttaacaattc cttttcaata ttagcaaaacagttaaggga 19080 caaaatgcag tggttcagtt atgtctaatt taacttattc aaaatattaaaacaaaacaa 19140 attgttttat ttcctttttt aaaagtcagg gtctcaatct gtctcccaggctctggaatg 19200 cagtggcaca atcatagcac actgtgttct caacctcctg gactcaagtgatccttccac 19260 ttaagcctcc tgagtagctg ggactacagg caccaccaca ccaagatcattttttaaaat 19320 ttttatttgc aggagacgag ttctccctat gttgcccagg ctggttttgaactcctgagc 19380 tcaagtgatc ctcccaaagt gctgggatta caggcatgag ccaccatggccagccttatt 19440 tcgtttctta ttaaagattt atattggcca ggcatggtag ctcacgcctgtaatcccagc 19500 actttgggag gctgaggcga gtggatcacg aggtcaggag atcgagaccatcctggccaa 19560 catggtgaaa ccccgtctct actaaaagta caaaaattag ctgcatgtgatggcgggcgc 19620 ctgtaatccc agctactcgg gagactgagg caggagaatt gcttgaacccagaaggcaga 19680 ggttgcagtg agccgacgtc gtgccactgc actctagcct ggcaacagagtaagactccg 19740 tctcaaaaaa aaaaaaaaat tatattgttg ccttctataa agtcatagtgattctcctct 19800 aagtgactta aatgttttaa tacttaaatt atcgtgcatg aaatttttcttgtccatatg 19860 ccacatgaac aaatatttgg cactaaggca ttaatcataa tagtagaaagatgtattata 19920 gccaaacaat tgccacttta gttggagtct tcttagacac aatatccaggaaatgctagt 19980 gaatcatttt gtgggtcaac ctttctacaa atttattctt tagattttctgtccattgct 20040 ttttttgtcc tttcctcacc ccgttttgtg ttggggggcc aagttggggaaggagattct 20100 ttccttcctt cttttccccc tattaaatga ttttgattga atgttagcttttgttaaaag 20160 agtatgtatg ttaagtatat ttcaaatgtt actagtttct aataggtgaatggtctcaat 20220 gactaaaaaa caaatatttt ttagaaacat tatgacctag agtatacattctgtaacttg 20280 agattttatg ctagtttgtc caacctctaa atacaccttg aatagatagtatatgtattt 20340 attcaaaacc attaaataat ggaatagata catgttaaaa attatgtatacaacatgatt 20400 ataactgcaa tatctatttt aaaacatgaa taaaaatttg aaggaatcttaaaatagtag 20460 tagattgtgg ttgcattttt attttattta tttatttttt agacagagtcttactctgtt 20520 gcccaggctg gagtgcaatg gcacattctc agctcactgc aacctccgcctcctgggttc 20580 aagcaattct cctgcctcag cctcccgagt agttgggatt acaggcacccatgcccagct 20640 agcgtttgta tttttagtgg agtcggggtt tcgccatgtt gatcaggctggtctcgaact 20700 cctgacctca ggtgatccac ccacctcggc ttcccgaagt gctgggatcacaggcgtgag 20760 ccaccacacc cagctgtggt tgcatttttt tggctcagtt ttcttttacaaatgaaacat 20820 catttttact actgttactg ttaatattct atgatgatta ataacatgccaaatatttct 20880 gcatatttca tattgatata atgtttaatg ctgatgattt ttattttatttatttatttt 20940 gaaacaggat ctcgctctgt cacccaggct ggagtgcagt ggtacgatcacagttcactg 21000 cagccttgac ctccctgggc tcaagcaatc ctcccacctc agcatcctgagtagctgaga 21060 ccaggagcat gcctggctaa tttttctact ttttgtagag acagagtttagctatgttgc 21120 ccaggctggt ctcaaactca tgggctcaag acatccaccc accttggcctttcaaagtgc 21180 tgggattata ggtttgagcc actgcaccca gacagatgat tgaattttagaaagaaaaaa 21240 gtaaatctat attgatccaa ttttggcttt ttaagtggaa atctcagagcagcaatgtgt 21300 ttaaagaaac ttcttttctg ctgttaggaa tgtcattttt atggtgttatagttggatag 21360 tatgccaaga gggggcatat ttcattttga ataacttgat ggatatataatttacatgcc 21420 ataagtcacc cattttaaaa tgtacacctc agtggttttt agtatattgccagagaggat 21480 gtacagccgt cacttcaatg taattttaga acatttcatc ctctcaaaaagaaaccccat 21540 actcattagc agtcactgcc cattagtccc tccccacagt ccttgccaaccactaatcta 21600 ctttctttct ctgtagattg tcggttctgg gtcggtcttt ctttctttctttctttcttt 21660 ctttctttct ttcttttttt tttttttttt tgacagagtc tcgctctgtcacctaggcta 21720 gagtgcagtg gcgcgatcgc ggctcactgc aacctctgcc tcctgagttcaagcagttct 21780 cctcagcctc ccaagtagct gggactacag gcgcctgcca ccatgcctggctaatttttg 21840 tatttttagt agaggtggga tttcaccatg ttggcccagc tggtctcaaactcctgactt 21900 caaatgatct gcctacctcg gcctcccaaa atgctaggat tacaggggtgagccactgca 21960 tccggccgga tatttcttat aaatggaatc atataccatg tggccttttgtgactggctt 22020 ctttttgcat aatgatttca aggttcattc atgttgtagc atgtatcagtatattcaggc 22080 actgcataac ttttcagtga attacagacc acatgtatga tggtggtcccataagattat 22140 aataccgtat ttttactgta cctttttatg tttagataca caaataccactgtattacag 22200 ttgcctacag tattcagtat agtaatgtga tgtacaggtt tgtagcccaggagtaataag 22260 ccataccata tagcttagat gtgtatatgg cttagatgtg tagtaggctctactatctag 22320 gtatgtgtaa gcacattcta tgatgttcac acaacaaaat gataatgcatttcctggaac 22380 atatccccat cgttaagtga tgcataactg tactttattc ctttttattgctgaataata 22440 ttccattgta tggatataat acattttgtt tatccatcat ttgatggacatttgggttgt 22500 tgccattttt gactactaca aataatgctg ctataaacat tcatgtacaggattttgtgt 22560 ggacatacat tttcatttat gttggatata tacctaagag tgatatcatatgataactct 22620 atatttaacc ttttgagaaa ctgccagact cttttccaaa gtggctgcaccagccagaca 22680 tggtggctca tgcctgtaat cccaacactt tgggatgctg aggtgaaaggatcacttgag 22740 tccaggaatt caagaccagc ctgagcaatg tagcaagaca tcatttctactaaaaagaaa 22800 aaaaaaaaga aggcagtgtc tgcaccattt tacattccca ctagcagtgtatgaagatgt 22860 tttcccatat tctcaccaat atgttattat ttgtcttttt aaaaattattttcatcctag 22920 tggatgtgaa gttgtatctc attgtcgttt tgacttgcat tttcctgatgacggatgttg 22980 aacatttttc atgtatttat tggtcatttg tatattttat ttggagaaatgtctatttag 23040 gtcctttgct aatttttttt ttttttttga gacagagtct tactctattgcccaggctgg 23100 agtgcagtgg cacgatctta gctcactgca acttccacct cccaggttcaagcgattctc 23160 ctgcctcagc ctcccaagta gcataccacc atgcctggct aatttttgtagtttttagta 23220 gagatggggt ttcactatat tggccaggct ggtctcgaac ctctgacctcaggtgattca 23280 cccgcctcac ccttccaaag tgctggggtt acaggcatga gccactgtgcctgcctgccc 23340 atttttaaat tgggtcattt gtctttttgt tattgagtca taggagttcattatatgttc 23400 tagataaaag tcccttatta gatatatggt tgcaaaattt ttctctgtttatacatgttg 23460 catcttcatt gtcttgatgg tgtcctttga agcacaaact tttaaaattttgatgatgtc 23520 taacttattt tttcttttgt cgcttgtgat tttgttgtca tatctatagaagggttctat 23580 attaaaatga tttagacaac atacttcaga aaacactacc agtaaaaaccaaatggtata 23640 gttttgagtg tttagtgatc ttggggaaac tattatacaa aatatgtcagctaataaata 23700 agttttattt tccttttagt cacttggaag ataggaaagt taacagatggtaattatttc 23760 acatctcaaa attcttttag agtggcatct aaatacaata ctaagtagaaattagccttt 23820 tgactaatat tcctataata tatttgaaac ttgaagatac tttcataaattaacaaatat 23880 ttacacacaa gggactagta cataaggtat tattaccaac atctatttgtgtagatataa 23940 attacaatag ctgtatagtg ttctattata tgaatgtacc ttgatttgtttactttaacc 24000 tgctccagtt tgttaggtta acttaaatct atttaaacca tttatctgaacttaccacat 24060 gtttatacca aagtactatg ctattggtac tagatttaca ggatatacggaaacttaaag 24120 atcatttttg aatcttctct atgttttcat taactgttta ctcctgttttgtgtcaggca 24180 ctgtgctagg ttctagcaat aatacaatag ataaaacatg atctctttaaagttttaaat 24240 tccatatgga gtgacaaatt ctgtgatatg cacattatgt actgtgatgaaagagtaatg 24300 attcctgcct ttgggtaagg aggaggatgt ggtgtatcac atggtatttgagcaaaataa 24360 ttgtatcata aaaggtacag taaaaatata tcatcttatg ggaccaccatcatatatgtg 24420 gtctgtaatt gactgaaaag ttatgcagca cctgcctgtg ctgatatatgctacaacatg 24480 aagttgtcca gtggacaggg agaaagaata atccacatag acagcatgagcaaaggtatg 24540 acatgttggt ataacagaaa caggccagta taaatagagt agctagagatgagtctgaag 24600 agttagctag ggagtaggtt atgaagaatc tgaaatccgt gctaaggcttgtgaattcag 24660 aaatagtaga gagccaatag aaccttttaa acaaatgaag aatatcggtatttaggacca 24720 tacttgatga cagtgtggtg aatgagagat gagaaagact tgagtcctggagaacatttg 24780 gaaggctgtt gtagtagtct aggttagcgg tcttcaaacc attgatcacacgtccccatt 24840 aattaaaatt tgtttgacca aaaaatacat atagatataa atgcacacacatttcaaaaa 24900 ctataatttg ttggcgtgta ctactgtttt aatatgtaac tgtagaaaaagatgagaaag 24960 ataattttga aatgaactat ttaaaaatac atttaaataa cttaaacttttcagattaat 25020 ggtacaaaaa accctgactg aatatgtgtc acacacttga actacagaaagttgcagtgt 25080 gctgaaaatg aatgaacgag aggtacactg ttaacttttt ttttttttttttaattgaga 25140 caggagtctc actctgtcac ccaggctgga gtgcagtggc gtgaacatggctcactgcag 25200 cctcaccctc ctgggcccga gcaatcctcc cacctcagcc ttcatagtagctgggactac 25260 aggcacatac caccatgccc ggctaatttt tgtatttttt atagaaatgggatttcgcca 25320 tgttgcccag gctggccttg aactccttgt ctcaagcgat ctgcccacctcaacttccca 25380 aagtgttgga attacaggcg tgagccactg tgtcagacct taactcttatgtgttgcaga 25440 actcccatta ctctcctagg attcctggtg taccgtgtgt tgcacctgaccatctaatgt 25500 ggaataactg aaggcaccag tatcaatcca tactcatgaa tattagtaaatcttaattcc 25560 taattgttca ctgaaaatta aatataaata tgtctaattt ttcctcatactgcagcatac 25620 cccttggcac ctctggtaca tatttttctg gattataggt aatatgttcaaagacacagt 25680 ggcaataatg atgatggagg gcatagatgg aagatttaga aaaagaatgaacaatttagg 25740 tatgtaagga aaggatagag ataagaatgg cttcatagtt cctagctggtgcattagtaa 25800 tggttttaac agaaatatag attctgaagg gaccaggttt ttgtatactattttgaatat 25860 ttggaggttg aattgcataa gaaacatcaa aatggcagaa gttcaataggttattggaaa 25920 taagtgtcta gctaagtgtc aggagataag tcaaaatgga gattgtgattcgtcagcatg 25980 taggtgatag ttaaaaactg gggaagtatg taaaaataaa agaactgagactagatctct 26040 gagatactat tatttcaggt ttaccagtga aaacacacag ggattggaaagataggagaa 26100 ccaggacaaa gtagaagcta tttcagagaa ggcaaggtaa ggatcatgattttctttttt 26160 tgtttttttt tttttttctt ttggagaggg agtctcgctc tgtcgcccaggttggaattc 26220 agtgacatga tcacgtgatc ttggctgact gcaacctccg ccttccagggtcaagcaatt 26280 ctgtctcagc ctcctgagta gctgggacta caggtgcatg ccaccatgcctggctaattt 26340 ttttgtattt ttagtagaga tagggtttca ctgtgatgcc cacgctggtctcaaactcct 26400 gagctcaggc agtccgccca cctcggcctc ccaaagttgc taggattacaggcatgagcc 26460 actgcacctg gccttaagga tcatgatttt caaggaacaa agtttgattacagtggcaaa 26520 tgctaaacaa agccaggcgt atagagactg aaaatgcgcc attacacatggttgttaaga 26580 agttggtggc ccagcctggt gcagtggctc acgcctgtaa tctcagccctttgggaggcc 26640 aaggtgggca gatcacttga acccaggagt ctgagaccag cctggccaacatggtgaaac 26700 cccatctcta ctaaaaatac aaaaattagc tgggcatcgt ggtacatgcctgtaatccca 26760 gcttggatta cttgggaggc tgaggtacga gaattgctta tacccaggaggtgaaggttg 26820 cagtgagctg agatcatacc actgcactcc agcctgggcg acagagcgagactgtctcaa 26880 aaacaaaaaa aagaagtcag tggcctgata aaaataattc acatatgaaatgactacaaa 26940 catagtgtat agtgaaatgc tctgtatgag ctgagctcag tggctcatgcctgtaatccc 27000 agcactttgc gaggctgacg caggcagatc acttgaggtc aggagtttgagaccagcctg 27060 gccaacatgg tgaaaccccg tctctactaa aaatgcaaaa attagctgggcgtggtggca 27120 cgtgcttgta atcccagcta ctcgggaggc tgaggcatga gaattgcttgaacctgggag 27180 gtgaaggttg cagtgagcag agattgtgcc actgcactcc agcctgggcaacagagtgtg 27240 attccatctc aaaaaaacaa aaatgctcag tatcgatttt atattacaaattattaaaat 27300 tttggccaag tgcagtggca catgtgaggc ctgtaattcc agcactttgggaggccaagg 27360 caggaggagt gctggaggcc agaatttcaa ggccagcctg ggcaacatagggagacccct 27420 tctgtatgaa aaatttaaag attagccagg tataatggtg tacacctatagtcctagcta 27480 ctcaggaggc tgaggtgaga ggattgctca cttcaggagt tggaggctgcagtgagccat 27540 gattgcacca ctgcactcca tcctctgggt gacagagcaa gatctgtatctttaaaaaaa 27600 gaaaaaagta ttaaaaattt gtcctggcca ggcgtggtgg ctcacgcctgtaatcccagc 27660 actttgggag gccaaggcgg gcggatcaca aggtctggag ttcgagatcagcctggccaa 27720 catggtgaaa tcccgtttct actaaaaata aaaaaattag ccaggcatggtggtgcgcgt 27780 ttgtaatccc agctacccag gaggctgagt ctgaagaatc acttgaacccaggaggtgga 27840 ggttgcagtg agccgagatc acaccactgc actccagtct aggcgacagaataagactcc 27900 ataataaaaa aaaaagaaaa atttttgttc tacccagcac tttgggaggctgaggtggac 27960 agatcacttg atctcaggag ttcaagacca gcctgggcaa catgatgaaactctgtctct 28020 acaagaaata caaaaattag ctgggcatgg tggcacactc ctgtagtcccagctactcgg 28080 gaggttgaga caggagaatg gcttgaggca gaggttgctg tgagctgagattgcaccact 28140 gcgctccagc ctgggcatca gagccagacc ttgtctcaaa aaaaaaaaaagaaaaaagat 28200 ctaaaatata atccctctct tctctgtttt gcatacctta aactttatcttgttgcacaa 28260 gtatttattg gctaccttct ctgctagtaa ccacagagta ataaagataagttagagatt 28320 ggaaggatac aaagagagac tgccagctgt tttagttgtg ttttataaacctctggatga 28380 ttttgacatt ttgttatact ttagcaatct tctttctgtc tatactgtagtgacacattc 28440 atttattgta gccatggata atgtcagtag acttttgggg aaaatattctttcatgttgt 28500 cttctgtaga ctagaataat atttttcatt ctgtcttttg ggagcagagaattaagaggt 28560 acctaataaa gtgagatgga ggtggatctc tataagctta tagtaattacaactcacagg 28620 aaaataattt gctctcctct tttttacatt aaagtttctc tcttcccatttttctgctgt 28680 ataagtcagg tggtaaaatg ggacttaatg aaattattat taaattttactttataatct 28740 gtgcaccaga gcatgatgga gtcaaaagag ttggtatcag aatgtaggaagtagtgattg 28800 aagtaagggt ggtaggatag gctgtacccc tttagaagat ctaatgtattcaggtagatt 28860 tcatttttga agttattacc aattattctt aaggattctt aaattctcacgtgacgttct 28920 aaaaaatgca tcacagtata attctgcaag aattcttttc ttctcagactaaggttatta 28980 gtgaaaggaa gccactaaag attggttaga cattcttatc tgtgtttactcagattttat 29040 ttcccaaatt actttcccaa gcactgtttt agaagttaaa atattttatgtatttttatt 29100 agtctagttt tactacttac ggtaagctaa gaacttgttt aacaatatacacttaaatat 29160 tttgctaaaa gtactgtatt tgaacaaaag attccactcc taaccctattgttgtaataa 29220 aagactagtg tcataaaata tagaggaaac tcagttatca gtttatgttaattaccagaa 29280 ttattcatta tttgtgttaa tttactatat ttgatgctaa aacattgtagtgtattcatt 29340 tgttatttgg aagacttcag tataattctt aaaatatatt tgtgagataattatgcttaa 29400 attttaatat aaaaatatta ttatacatat ttgttttttt aatcttcaaattattttcac 29460 taatattcca tttggttcct gagactgtta tctcattttt attgataaagaaactcaagt 29520 cagagaagtt aaataacttt cttaacaact tagtgacaga atgggactaaaaactcatgc 29580 cttcttattt cggtacttat attatcatat attttaaagg tttctcaggttggtagtttc 29640 ccaattccaa gtttcatcgt aatataatag cacctttgct actatagctgactagatggc 29700 ttaggaaact agataaatta ctgttctaaa gagtgttttt tctctagctccacatgccta 29760 cctattaaag attctaataa actacccttt tccctaatat cctttgagataagaaatgag 29820 aatttactgt ccctagattt gccattttgt tagcttgcat actaaaatctgctggatgcc 29880 catattccca gttactcaga aggctaaggc aggaggatca cttgaactcaggagttttag 29940 gttacagtga actgtgatca catcactgca ctcaagtctg ggcaacagagcaagacctgg 30000 tctcaaaaaa aaattttttt ttttcgctaa aattctaaat atatgaatttggccaggcac 30060 agtggctcac acatgtaatc tcaacactct gggaggctga gacaggaggattgcttgaac 30120 ccaggagttg gagaccagca tgggcaacat agtgagaccc catctctttaaaaaaaattc 30180 taaatatatg aattaattgt gtcatattag tgagaggtta aaaaataatataactcttgg 30240 ccctcatgaa agatgactct ctttgtagca taggttttgt cagttatgaacttaaaaaac 30300 tgtcaagtgg aaaaattgac ccgtatacta tagaattcca gtgctatttccttaaggctc 30360 tgaactaaat tgtcaaatgt aagtgtaatt attcataaaa gtaatatctaatgccatgat 30420 acgatttacc atgataaaat ttacaatgtt tcttttcttt ttttttttttatttttattt 30480 ttttttgaga tggagtctcg ctctgatgcc cagggtggag tgcagtggtgcgatcttggc 30540 tcactgcaag ctccacctcc cgggttcaca ccattctcct gcctcaggctcccgagtagc 30600 tgggactacg ggcacccgcc accaagccct gctaattttt tttttttttgtatttttagt 30660 tgagacaggg tttcaccgtg ttagccagga tggtctcaat ctcctgacctcttgatccgc 30720 ctgcctcggc cccccaaagt ggtgggatta cacgtgtgag ccactgcgcctggccattgt 30780 ttggtatttt ttaaattaaa ttttactata agagggtatc gcggatatttcattttaatc 30840 ttaaatttat atcttccctg acagttggtc atatagctgc tctttgaacagttctagcaa 30900 agatgaactc aggtacttta taaggtagtg taatttactt ttgaataactctggtggtta 30960 gaaaatcctg aaaatttcgt tccaaaatgt ctaatgttcc aactatttttcctagttctt 31020 tctactcagt ttttcacatg acaatctatc aaaatagcca aaaacttattctgtaatttt 31080 ctttgagttt ttcctattcc agactaagac tttcctattc caaaccagacttggtttgat 31140 ttatttttac tcatattaca tgatttccta tcaggattgc ctccttctcattatatttga 31200 atttttaggg tcttaaattt ttcacaaatt gaacagagta ctccaagatgtaaaatgatc 31260 tgttttaata tatggtggga tagttacctg ccttattaag tggattatctgagtacttaa 31320 atatatgcaa cctcagtttt ttacttttga ggaagctaca tcatactgttagctcatttg 31380 gggcttgtac ccttttccac agcagcagtg ggtaagttga gattctctaatttgttctta 31440 ttacagttga agtgtttgga cgttaagtgt ttaggtatta cagttacccttattatgttt 31500 cttctttatt ttgttcattg ttgtagttgt tagtagcttt taacatagtaatttatgttt 31560 ttaacatgct agtcatgtct tccaagtatg tgttgttaga ggtttcttaaccaacagata 31620 tttattaatt acttactata tgtagggcac tagatatgtt tcctttccatatggtattga 31680 tagttttcta aggtggaaaa ttattccagt gttcaagtat acttagaaaaagtagggaaa 31740 cttaccctgc cccataataa acactctagt gaaaacatac gttactggccaggcgccgtg 31800 gctcacacct gtaatcccag cactttggga ggccaaggcg ggtggatcacgaggtcagga 31860 gatcgagacc atcctggcta acacggtgaa accccatctc tactaaaaatacaaaaaatt 31920 agccaggcat ggtggcgggt gcctgtagtc ccagctactc gggaggctgaggcaggagaa 31980 tagtgtgaac ccaggagggg gagcttgcag tgagccgaga tcacgccactgcactccagc 32040 ctgggtgaca gagcgagact ccctctcaaa aaaaaaaaaa aagagaaagaaagaaaacat 32100 cttgtactgg cacaagaaca gacaaacaga acactgggac agtatagagaactcagattg 32160 atttatatat gggaagtgaa tgagataaaa atgccattac aaattgattagttaagtata 32220 agccgtttag taggtggcat gaagaaacct gctcactaga tggagataaataaaattggg 32280 tccttgctgt ttataaagaa gaaccaaaaa atgtagacaa aactgtaaagttaataaaac 32340 ataatttatc tttttgactt tgtattagga aagtatttct taatacctcaaaagcttgaa 32400 gtcataaggt gaaatttaat ttgatgatgt tctcaaattc attagtaataagagaaatct 32460 aaattaaaac atgcttttag tttataccga ttaggctaac aaaaattagaaagcagaact 32520 ggataatgcc acatattggc tggacgcagg gaaataggag cctcaaggagtgcaggttag 32580 tgtagtcatt cttgagaaca atttggcagt acatcattta cttgataagattcattttga 32640 cctaacagtc tgtttctgtg tctgtaagca gagattaatt ctcacatgggttcataaggg 32700 gacatgtaag gagataattc atttcaacat tgtttttggg tgttgcaattaaagatactg 32760 tgaatctgca atgctggggg agtggataga caacatgtgg agtactttgcatcaatcaga 32820 aacaatgaac caagtcactg aaattgtata caaccaattt tttacagataaatcttttaa 32880 aaataataaa aataaatata ccataataaa tataccacag caacgtgaataggttttaaa 32940 acatctaatg cagagggtaa aagtaggtat acatagaaat agaagaatgttcatcaaaca 33000 aattagaaga ataacttctt gggaagggga atggggttag gaaatggagatggaaagaaa 33060 atcagttaga agagggatat tatacagatg atgataacac accatgaactgaggagtatg 33120 attaaatttt ttgtacccaa ggaataaaaa tggggcttgg gaaattatttaacagtgaat 33180 gtaaattggg ttggagtaaa aaaaaaacta tgaacataaa aaagaaaaacttaaaataat 33240 ccaggtacaa ggacatgagg cttttcttta ggattgtggt aatcgggaatggaaagtaaa 33300 ggactagtat aagaaatact gcaaagacgt ggtgtcaaca accagttgggtgaagagatg 33360 agccaagttt tgaatctggg taactgggaa gatggagata ctattaattaaagaggagta 33420 gcaaacagaa gataaccaca tgtaactctg acaagatgtg ttgaattcagctctagatat 33480 attccattgg aagttccagc atttcactga ggtagttatt cactgttagagcatttattg 33540 aggatatact atgtgttagg catcaaagca ggatatatat atatattttgccaaaattgt 33600 agcaaaaatt gtatatgtgt atgtatatat cctgctttga tatataagtatacatttata 33660 tatgaacata tatatgtatt taagttcaga ggtatatgta caggtttgttatataggtaa 33720 atttgtgtca tgggtttatt gtcacccagg tattaagcct agtacccattagttattttt 33780 cctgatactc tccctcctcc taccctctac cctctggtag accccagtgtctatagttct 33840 cctctatggg tccatatgtt ctcatcattt agctcccact tattaagtgagagcatgcag 33900 tatttgggtt tctattcctg tgttagtttg ctaaggataa cggcctctagctccatacat 33960 gttctgcaga gtacatgatc ttgttctttt ttatgactgc atagtattccatggtgtata 34020 tgtaccacat tttctttatc cagtgtacca ttgatgggca tttaggttgattccatgtct 34080 ttgctattgt gaatagtgct gcagtgaaca tacacatgca tgtgtctttatgatagaaca 34140 atttatattt ctttggatat atacctagta atgggattgc tgggttaaatggtagttctc 34200 tttttacgtc tttgaggaat tgccacactg ctttccacaa tggttgaactaatttacact 34260 ctcaccaaca gtgtataaac attccatttt ctccacaacc ttgccaacgtctgttatttt 34320 ttgacttttt aatagtagcc attctgactg gcatgagatg gtatctcattgtggttttga 34380 ttcgtatttc tctaatgatc aataatattg agcatttttt catatgcttgttggctgcat 34440 gtatgtcttc ttttgaagtg tctgttcatg tcatttgccc acttgttaatgggttttttt 34500 tttcttgtaa ctctgtttaa gttaaagcag gatatttttt attcctaagatgttttggcc 34560 ctggtatttc agtctcctcc attttgatcc ttagagtgat ttgattgggtttcccagaat 34620 tcgtaaggtt gaaattatac cagtttactg ttgagttaaa aaaaaaaatgacaaaatggt 34680 taaaacatcc caacaaatta gatctgtaaa atttcaccta agaaaccagattttggctgg 34740 gcgcagtggc tcatgcctct aatcccagca ctttaggagg ccgaggcgggcggatcacca 34800 ggtcaggagt tcgagaccag cctggccaac atggtgaaac cctgtctctactaaaaatac 34860 aaaacttagc tggacgtgat ggtgcatgcc tgtaatctca gctacttgggaggctgaggc 34920 aggagaattg ctggaacctg ggaggcggag gtagcagtga gccgagatcgcaccactgca 34980 ctccatcctg cgtgacagag caagacgctg tctcaaaaaa aaaaaaaaaaaaagaaaaag 35040 aaagaaacca gatttagtaa tagtggccac aaaggaaaac ttaaaatggcagagaatcat 35100 tgaaatttgc tttgaactaa aaaatgaata ggtgaatcaa atttttgttgtaaaatttac 35160 tctagaagag gcttgatact gttttcagta gtttcagtaa aacaaaattgaaaagggagg 35220 gaaaaaatag aaactttgtg ccatgtagaa aatcgctctg ggtatccagtcagcttgatg 35280 tttttattgt tccagaaaat gctgggttct tgcccccact taactgaggtagacttgaat 35340 ctctcttttt tttttttttt tttttttgag acagagtttt gctcttgttgcccaggctgg 35400 agttcaatgg cgtgatctcg gctcaccaca acctctgcct tctgggttcaagcaattctc 35460 ctgcctcagc ctcccaagta gctgagacta caggcatgcg ccactgtacccggctaattt 35520 tgaattttta gtagagacag ggtttctcca tgttggtcag ggtggtctcaaactcccgac 35580 ctcaggtgat ccacctgcct tggcctccca aagtgctggg attacaggcgtaagccactg 35640 tgcctggcct tgaatctttt aaattagttt tagattatat caatactatgtgagtacatt 35700 ctcattgtaa tatatttaga ttttataaac aaaataaaag tgaccctgtaatcctgtcac 35760 ttccctctct agaggtaacc accatttgga atatatcctt tcagtttctctgcttttaca 35820 aatgtgtgta caaaaaatac gtattgattt gtgaaaattt ggtatttcctatatagtctg 35880 taagttttga atatataagt attgaatatc tttctgtgtc agttacattggctcagatgt 35940 ttgctgggta aaaatggagg ttttataata tccttactag ctaggtactgtaaggatggg 36000 aggagaaagg aacacaggta tgtttgctgc tctcaggagg ctcacagttagaagaattga 36060 aaatatataa tcattataat agacaaagtg tcctaataga gatatgtactaggtgtcttc 36120 ttggtacaga ggaaagatta ctcttggaga aagaagttaa tatgttagggagtgcttaga 36180 gatggtaaaa gagattttga atcactttct aagaatattt tgcaggtcagatgtatcctg 36240 gaatgattaa gtaatatgct cgcttcattc ttctctgtct agtgaaatgtatgtcatctt 36300 ttggcaataa tcaatctttt tggtgtctta agccaagatt ctaaaagcaaaatctttatc 36360 atatatgaat attttttaga attttgacag ctttatacag tggcagaaattacttctgtt 36420 aatattttta tatttcatct tacagaggat tatgtgaata tattgtttcccttctttagg 36480 gaggtggttg actcaatggt tcagcatttt aaagtaacta tatttggagaccgtagacca 36540 gtttatgatg gaaaaagaag tctttacacc gccaatccac ttcctgtggcaactacaggg 36600 gtaagatatg cattcctgta ttggaaaggt atatttttga agtgtctccttttacacgca 36660 tttattacca tttttattac agtccatata tatgtgaata tttatcactgattgttttta 36720 actttttgtt ttgaaataat ttcaaactta aagaaaagtt gcaggaatcatgcagagaac 36780 tctcatacac cctttatgta gcttcactga ggttctgaac atttccacctttgttttatt 36840 ttttttcttt ctctcttgta catacatact tattttttcc tgaaccattcacaagtaggt 36900 tgcacatacc atgccccatt aatatttatt ttattttatt ttattttattttattttgag 36960 acagggtctc gctttgtcac ctaggttgga gtgcagtagt gtgatctcggctcactgcaa 37020 cctctgcctc ccagattcaa gtgattcttg tacctcagcg tcctgagtagctgggattac 37080 aggcacgtgc taccactctt ggctaatttt tttgtgtttg tagtagagatggggtttcgc 37140 catgttggcc agactggtct caaacttctg acctcaagta atccacccagctcaacctcc 37200 caaagttctg ggattttggg agtaccactg cactctgcca gtatttaatactttaatgta 37260 tattcctaag aacaatgata aaaacccttg tacagttatc aagttcattaaatttaacat 37320 tgatatgata cttttattta atcaacaata caaattccag ttttaccagtttttccaatg 37380 atgcccttta gtattatttt tctcctctgt tacagaatcc agtccaggatcatgatatag 37440 ataccatgtc gttctttccc cagccttttt ttatccttca tgacagtaacgtagttgaag 37500 attatcggta aattattttg tagaatgtcc ttcagtctgg gtttgtctgatacttcccct 37560 tgattacatt ctggttatgc attattggca ggaatattat ataaccattctttccttatc 37620 agtgcatcat atcaggaagc acgcagtatg tatttgttcc attattggtgattattggtg 37680 atgttaactt tggtcagttg attaaattgg tgtctgccag ttttctcctattggggtatt 37740 cttttcctcc ttgtagttaa taagcatctt gtagggagat tctttttgtaattgtgataa 37800 aacatatgta acataaaatt taccgtctta accattttta aggtatatttcagtggtatt 37860 aagcacattt acattgttat ataattatta ccaccttcca tccccagaattgtctttatc 37920 tttcaaaact gaaactccat atccattaaa cagtatccat tgctccctcccctcagccct 37980 ggcaaccacc atagtacttt ctgtctctga atttgactat tctaggtacctcatgtaagt 38040 gaaatcatag attgtttgtc tttttatgac tggcttattt cacaatttatccacattgtt 38100 tcatgtgtta gaatctcctt ttcaaggctg aataatattc cattgtatgtatgtaacaca 38160 ttttgttaat cccttcatgc atcaatggac accttttggc tattgcaaataatcctgcta 38220 tgaacataga tgtaaaagta ttgaactctg ctttcggttc tttggatatatacccagaag 38280 taaatttgcc aggtcatgtg ataattatta attttctgag aatctgctgtattgttttcc 38340 agaatggctc catcatttta cattcccacc aacagtgaac aagagttccaaactctcact 38400 tccatgccaa cacttgtttt ctgttttgtt tttttttttg ttcgttttctaatagtaacc 38460 atcctaatgg gcgtgagatg atatctcatt gtggttttga tttgcatgtcccccaacaat 38520 tagtgatgtt gagtataact ttgtaggctt attggccatt tgtatatcttctttggagaa 38580 atctgtggtc aagttgtttg cccatttttt gaactgggtt gtttgttttgggttttgggc 38640 agtgagttgt aggagttctt tatttttaga ttttttattt tttttagctaaactgatcaa 38700 taccattgta ggagttcttt atatatattc tggatattaa cttctgtattctggatatta 38760 actatatata ttctggacat taacttctta tcagatatat aatttgcaaacattttctta 38820 catttcacag gttgtctttt cactatgttg tgtccgttga tacacagaagtttttaattt 38880 tgaaatggga gatactttta aggatcctgt tgctcattgg attttgattattcttgcttg 38940 gcaattgatt attcttgctt gaaaggatta ttactatgta gaagtggtgattttctaatt 39000 ctgtcattca ttctccgtat gttcattaat ctgctgtaag ggagtatattttcttttact 39060 ctatttattg atttcactgc ccagattgtc ccagatttgg ccagtgggaactcttttaag 39120 ctgactcctg tgtccttttg aaatgacact ttttggggaa tacaatcctgcatcacctaa 39180 aacaatgggg atatgctctg cgaaatgtgt ccttgggcaa ttttgtcattgtgctatcat 39240 cacagtgtat acttacgcaa acctgaatgg tatagcttac tacacacatacaccatataa 39300 tatggtttat tattgctata aacctatata ggatgttact ataccgaatactgtaggcac 39360 ttgtaagaca atggtaaata tttgtgtatc taaacgtatc taactatagaaaaggtacag 39420 taaaaataca ttgtaaaaga ttgtttaaat ggtacgcctg tataaggcagcttcattata 39480 atctaatggg accaccgtgt atatggggtc cattgtggac caaaaatcattatgtggtgc 39540 atgactctac ttcattactt tctggcacag gatgttcctg gtttattttatgttttctct 39600 gctgcagtcc tgcaactaac catttctctg aggatctctg gtttcttttagttggcaaat 39660 gcaattttga aatgaaaatc tgggtactgg tatgttcaca gctgctggggtatatgtgct 39720 tctaagccct ttcaatggat agaggtagga aatttataaa taagtagataaataaataaa 39780 acggacaata aaaacatatt acactttgta tggtaaatct aacacatatatataggaaat 39840 cgtgagttca cacccgattc ttccaattct agtccgtgcc tcatgggattcttcctttcc 39900 tcactccatt tcatatttgt atctttcttc tttagttaga ttcctggctcctcaaaacat 39960 caccactcct tctcatttgc tcagtcctac agtacatata aaatagtttgtgagaaaaca 40020 aacctactaa tgattcaagg ctttattttg tatgcaattc ttctaaatccaccctttctc 40080 tacccctaga attaagacta ttgtcagtat acatactgtg ttcaagagttacttgaatta 40140 gagcttcctt tttctttttt ttcaatgtgg ttatgttatt tatttgaaatttatttgggt 40200 tcatttgatt ctgtttatga tattctgttt taattttttc cctccctccctttgtttatt 40260 tatttattta tttagacagg gtcttgctct gtaacccagg ctggagttcattggcacact 40320 cacagctcac tgcagcctca acctctcagg ctcaaacgat cctactgcctcagcctctca 40380 agcagctagg accacaggtg tgcaccacca cacctggcta atttaattttttgtagagac 40440 taggtcttgc tctgttgcct gggttggttt aaaattccct ggctcaagcagtcctcctgc 40500 ctcagcgtcc caaagtgctg ggattacagg agtaagccac catacccagcctaaatatat 40560 atatatatat tttttattat aaatatataa attataaata atatataaatttatattata 40620 aattataaat aatatataaa tttatattat aaattataaa tttataaatatataatatat 40680 atttatttta tttatttatt ttttttttga gcctcactct gttgcccacgctggagcaca 40740 gtggtgtgat cttggctcac tgcaccctct gcctcctgag ttcaaatgattctcgtgcct 40800 cagcctcccg agtagctggg attacaggca tgcaccacca cacccagctaattttgtatt 40860 tttagtagag acagggtttc accatgttgg ccaagctggt cttgaactcctgacctcaag 40920 tgatcagcct gcctcagccc cccaaagtgc tgggattaca tgcatgagccactgcgcctg 40980 gccttttttt tttctttttt ttaatatgta gaactttaat atgcttccaaatttcaaaag 41040 tataccaaaa catatactca gaattgttct gtccttattt tttccagtccattccctccc 41100 atcctttgaa agtaactagt ttctttgttt ctggttcatg cttcctgtgtttctttttgc 41160 agaagtaagc agatatgtga atattttcct cctttcttac acaaaagatgtcataatatt 41220 tgtaatcttt tgtactttgc ttttgtcact taatagtata gcttggaaatttattccatg 41280 gcagtttcaa gagattttcc tcattctttt ttcatagccg catagatgttggagcattta 41340 gggtagtttc cagtattttg caatgacaca taatgctggc acgagtaactatgttattta 41400 aattttatct acaaaaaaaa atggtatatt ttagtctatt gactttatttttgccatgtt 41460 ttctcttaga tatttttata agttaaatct aaaataattg tatttatctttttcaacatt 41520 taccagttga ctcttttccc atcaacaggt agatttagac gttactttacctggggaagg 41580 tggaaaagat cgacctttca aggtgtcaat caaatttgtc tctcgggtgagttggcacct 41640 actgcatgaa gtactgacag gacggacctt gcctgagcca ctggaattagacaagccaat 41700 cagcactaac cctgtccatg ccgttgatgt ggtgctacga catctgccctccatgaagtg 41760 ggtgcttctg ctttttttct ctttagattt taaactccca agaatgaattgtgcaggctt 41820 cccttggtta aacctttatt tgtcatatat tttgattgtt caactgaaatgttgaacaag 41880 aatagcatcc atacaaattc attgacagga gtacgttaca gaaaattatctggcttttgc 41940 aagtaactat acgtcattag cttagctagt ctcatgaata attttatagaaaaatatctc 42000 accctttctc ttaggatcta aaagtcttaa cagatctatt ttcagatgtatttatttagt 42060 tatcttgttt taaaagtaat ttcactgttt atacaacaat atcaaattgtgttgaattgc 42120 tttttttcaa taaccctagg acctcacatg tgtagggtat gctcctgtgtgtgagcgcat 42180 gtgtacccgt gtatttttta ttgtttggtt gggttttttt tgagacatggtctcactctg 42240 tcacccaggc tgtagtgcag tggcacaatc atggttcact gcagcctcaacctcccaggc 42300 ctaagcaatc ctcctacctc agccttctga gtagctggga ccacaggtatgcaccatcat 42360 gcctgactaa ttaaaaaaaa attttttttt tttttttttt ttgtagagatggaatctccc 42420 tatgttgccc aggctggttt caaactcctg gactcaagta atcctctcaccttagcctcc 42480 caaagtgctc ggattacagg tgtgagcccc cacacctgac tcagtatgtttttttttaaa 42540 gaaaaatagt atgtcttgca aacacattta tataaatacc tttttgttcaataattattt 42600 acttgttaac atttttaagg tcggaactgt taacttttta aaacctatttttaagaaatt 42660 attttaaata aaatttattc ttatttcaac caacaatttt gagaaaggaaaatttaagta 42720 gatttttttc catttagagt ggatactttt tgctttctca aatttggaacatgtttagtt 42780 tcatatattc ataatgataa gcatcattat gttaattgtg ctctagtctccccttttctg 42840 cagatttaaa tacttgcatg agaaggaaag gattgaacat gccattttaatttttgtaga 42900 tacacacctg tggggcgttc atttttctcc gctccagaag gatatgaccaccctctggga 42960 gggggcaggg aagtgtggtt tggattccat cagtctgttc ggcctgccatgtggaaaatg 43020 atgcttaata tcgatggtaa gggaactaaa gccatattct gtattgggtggtggatttct 43080 gtatgatgtg tgtacataaa ttttatatat aattatacat actggtgtctcgaagtaata 43140 tttggacatg tattatgatc tactggagaa acctttatat ttttattacatttcatttag 43200 aaagcctgta gaatttacct tggaatgctg ctaaacatga agcaagcacatgaagacaga 43260 tttaaaagcc ctgatgatta tctgagcaat cttctattat aactcacttttgccctttta 43320 actctaagcc aacattttat tatgaaatat atattttaag aaagataattctgttgggca 43380 tggtgacccc cagatgttat acccactgct ggtcttaatg tgatgctaattgcattcttg 43440 tttttaggtg ttttcttaca aaatattttc aagcttatgt aaaaacagagagagtagtat 43500 tatgaactac cacataggta gccattactc agattgtcag aatttttctacctttgcgta 43560 atccgagtac atttcttcct ctacagtagt gtttttaaat ctaattccagacagcatgtt 43620 attttatccc tattacttca atgtgtacct ctaaaactat ggatattttcttatagcagc 43680 aatggcatta tcatatgtag gaaaattata aacaagcatt tatttttaccctctaatacc 43740 tgtccacaat cagatttccc tgattgtctg aaatatgcct ttttcttgttagctggttct 43800 aatcagaatc caaacaagat ccacacatca catttgatta ttgtgccttttgccatctag 43860 tagtcccatt tcctttcctg ttttcttatt tctttatgcc attgacattttacaaaaact 43920 gggtcacttg ttctgtagaa tatgttcaaa tctgattttg tctttttgttttcttgtgtt 43980 attaccttgt tcctctatcc cctgtttttt ctgaaaatga aagttagcttagaagtttca 44040 ttccattctg gttcaaaatg cttaagtgct ttatgtcgtg tcatattaggaaacacagta 44100 tctagtggtc ccaattttag tgattcaaaa atcagtctct aggttcagagattaatcagt 44160 agattcagag atctctccat tgtaaatttc ttaattaacc tttgaattgctaatgttctg 44220 ttcactgatc gttgtggccc aaattattta tttcactagg gattacgaactggtaatttt 44280 tctgttattc tttttgcatt aggtggaatt tttctgtgga aaagctcagtgtcctattga 44340 aaattagtaa atgtttgaaa gctttcttgc tttcgggcac agcaggatattccttgctca 44400 tcttatattt cctgcccagt acctgaaatt agacattcct ccaaggatccctggatcctt 44460 ccagattagg gtatagtttc ctcatttttt cagctgcacc taccattgtgtaatatatga 44520 ttgaggaggg gatatattta accagttttc tttagatgta tactagggttatttttaact 44580 tttctatttt acatcatttt atgtatatta agataagtct gtaagatacattcctggcgg 44640 ttagactgat gagttaaggg caaatgtatt aatatttgta atttgtatcattgttgccag 44700 attgcactcc atagagatag taaatatttt gcatcaatga atgagagtaccagtttcctt 44760 atatctttgc cactagaatc atcaaactct tagattttct tcaatcagatttgcaagaaa 44820 cagttttctt gtgtcttctt aatttgtatt tgactacctt ttcacaaatataagggccac 44880 ttgtgtttct tttttaatga actgttagtt tatatctttt gcccttttttttctattgga 44940 tttttcgtct tcttatttac tgagaatgct ttctaaatta aggaagttagctgttcatct 45000 gtcatgagtt gcagatattt tacctaggtt gctatttgtc ttttgacctttggtattgtg 45060 atgttttttt tgttgttgtt tttgtttttg ccatgcagaa gtttgtttgtatgttggtgt 45120 gtgcacacat acatatgtat tggtcagtca tttcttttat gacttttaatatctgatagg 45180 actagtccca tttttctctt ttcagggttt tccagaccat tcttatgtataagctttaga 45240 aattccttgt ttagatccag tgagtgggag aggacagatt gttatttttattgagatcct 45300 attagattta taaattaagt taggacaaat tggcatattt aaggtgttgtcttttctgtc 45360 aaagaatata ctatgtcttt ctatttgttt aagattactt tcattttcaagagatcttaa 45420 agcttcacat aaattttgca catttctttt tttttttctt tctttcttttcttttttctt 45480 tctttttttt gagacagagt ttcgctctgg agtacaatgg agtgatctcggctcacagca 45540 acctccgcct cccaggttca agcaattctc atgcctcagc ctcccaagcaactgtgatta 45600 caggcatgca ccaccacacc cagctaattt tgtattttta gtagagatggggtttcacca 45660 tgttggtcag gctggtctcg aactcctgat ttcaggtggt ccacccacctcagcctccca 45720 gagtgctggg attacaggcg tgagccactg cacccaggcc atttcttgttattccaggat 45780 attttctcct ttttgttgct attaaatggg tttatagata ctaactggtaattgcttata 45840 tatattcagg cttttgattt ctgtatactt tctctctttc tgtgttattgaattccatta 45900 ttgtttgtgg tattttttca agcgatttta ttggggtttc taggttataatcatcacctg 45960 caactagtga taagtttacc tcttcctttg gatttttata cctctaattttttttatcag 46020 attatataga ctaattagac agggtaggat taaatagtag tgattaatgtgcgtcctttt 46080 tctttcttgc tctagaagca ttaagcattg tatgtataca ttgtcatattaaggaatatc 46140 tgtttcttat taagtacgtt tatcaagagt atgtgttaaa ttttgtcagatgccttttca 46200 gcatctatgg agctgatgat gtgatatttc tcttattttt aacaaacattgaaccaaacc 46260 aggactccta gattctttta attgatgctg gattctgttt gctaatactttccttttata 46320 aattaacatg cagaagtcat attaatctgc agccttcctt ttttgtacaatctttgtcaa 46380 gttttggtat tatttcctaa aaaattcaga agttttccat tttctaaaatgtataacagt 46440 tttaaaatag tattgatatt atcagctctt gtatgatttc gtagaattcccctataaaac 46500 cttgtgacct gccgtttttt gtttggtttg gtttggtttg ctaggtagctgttttaaaac 46560 ttccctcatt tattttgtga aaatcagtct aataatttat cgtacaattaaaaataacta 46620 aaaatataat tggattgttg gtaacataaa gaaaggataa atgcttgaggtaatggatac 46680 cccatttacc ctgatctgat tattatgcat tatatgcttg tatcaaaagctcgtgtaacc 46740 catagatata tacatctagt atgtacccat aaaaattttt ttttaattaaaaaaaaaagt 46800 ctgttcaggc tgtttaggtt cacttttggt agctcacatt ttactggaaaattattttat 46860 tcatatgtaa tttaattata cagagtttta caaagtagtt tctatcgttattatgatttt 46920 tttttttttt tttttgagac agggtcttgc tctgtcaccc aggctggagtgcagtggtgt 46980 aatcttggct cactgcaacc tccacctccc agggtcaagc gatcttcccactacagcctc 47040 ctgagcagct gggactacag acacatgtta ccacacctgg ctaatttttgtattttttgt 47100 agagacaaga tttcaccatg ttgcccaggc tggtctcaaa ctcctggtctcaagagatct 47160 gcctgccttg gcctcccaaa gtgctgggat tacaggcatg tgccaccgtgcccagctagt 47220 ttctattagt ttttgaaatt ctgtgtattt ttgtggtttt cccagtctgttattttgcat 47280 atgtatgctt tgttcttttt actttttttt caggtagtga aattcagtggtatttatttt 47340 tctccatctt tttcaagctt tcaacaataa gcatctatta cttcttatcaaaaaactaaa 47400 aaaaagacgg tatggcatca taaatattta ttttagaaaa caaaatgtatttattgagtg 47460 ctttctgatt aagaacaaag tatagttaat gaatgctgat tgtttaattaagtagtttaa 47520 tccttaacct tttttcctat gccctgactt cttttttggt aacatagtcttcccaattaa 47580 tgaattactg aaacctataa taaagaatat tttctattat tctagctcaggtgtatattt 47640 agtacttaaa cttaaatact tgaatcaatg aaataaaatc ttgatgaactctttctagag 47700 atgtaaggta cccaaatttc ttgacacaat tttttttgag ttttgctcttgtcacccagg 47760 ctggagtgta gtggctcgat ctcgactcac tgcaacctcc gcctccctggttcaagcgat 47820 tgtcctgcct cagcctccca agtagctggg attacaggtg catgccacgacacccagcta 47880 atttttttgt atttttagta gagatggggt ttcaccatgt tggtcaggctggtctcgaac 47940 tcctgacctc aggtgatccg cctccctccg ccttcccaag tgctgggattacaggcgtga 48000 gccaccgcat ctggcctgaa aacaattttt ttttttatta acgttaaactcactagaaaa 48060 ctctcaagaa ttgtgttgaa gatcatttga aaatatatct gccaacttcctcttccttcc 48120 tgaagtgtgt ttacagacag agcaagttac aacagtctta ctattctttgaggatagagc 48180 agcttcctgg ggattctgga ggctcagttt tctggtctgt tattaggacacaatactgat 48240 gttgaggaaa gtaaggcctt gctgacaatg ggaattattc ttaaaagttatttttctatc 48300 tattttagct agattagtaa catgtacttt cattttgttg gtggaaatatttaaaacaat 48360 tatttttctg gtgatcactg ttttagccgt attacaaagc ttctaatatgtagtattttt 48420 attatattct agaaactaga aatacataat attattttct agaaactccacaaatttcat 48480 ttgtatttcc tctgtgcttg aaatattgtt tgagagactt ttcaaattttcagattgaag 48540 atctttttgt tttctggatt tgttgtgaat ttctagtttc attgtgttgttaaaaaatgt 48600 tgtttgtggc tgggcgcggt ggctcatgcc tgtaatccca gcactttgggaggccgaggc 48660 gggcggatca cgaggtcagg agattgagac catcctggct aacacggtgaaaccccatct 48720 ctactaaaaa tacaaaaaat tagccgggcg tggtggcggg catctgtagtcccagctact 48780 tgggaggctg aggcaggaga atggcgtgaa cccaggaggc ggagcttgcagtgagccgag 48840 atcgcgccgc tgcaccccag ccttgggaca gaatgagact ccttctcagaaaaaaaaaaa 48900 tgtggtttgt atttttcctt ttagagtttc tagaggtatt ttttgtagcccaatatacgg 48960 tcaatttttg tgatagttct atggctatat gaaaataaga tatattcatatattagttta 49020 gagtgaaata tccatcagag ctaccagttt gattatgttg cttatgtcatttatttcctt 49080 agttttgttt tttgttctct cagactagga taaataaatt tcctattattaatatctttc 49140 tgtttcttct tttatctcct gtaatttcca ttttctgaat gttgctactgtattaggagc 49200 atatatattc atatctgttt tcgttatgaa ttataacctt tatataagtgactttctttt 49260 tgtacttttt gaccaaaatt ctacattatc taataaaaag attgcacttcatgcttttgt 49320 gaactttatc ctatccttta actttttttt tcaggtatat cttgatgacagcatagaatg 49380 gtttgctttg tgggtcagtt taaagctttt tttctgtttg gtggatgaattaagccagat 49440 atagatagga aggacatatc tttctgtcaa gtcacaacca ttggtcatggttttgtaaaa 49500 ttattttatg ccatttttta catttgtata tattttgaaa ctttgagtgttgtgttttct 49560 gtgcttttta aatggtagtc ctcatcttag gaagattttt ccccccagtggttatcttta 49620 tacttatacc ttcatatgat acctatcttc ctctgtttta aagcagtcttttgtttccct 49680 taattgagta acaattacat tagctttatt ctcttccctt ctttctactagttttagcca 49740 atatagtatt attttattgc tctttataat tttattctgt catgttgcttaagtttttac 49800 tgattgactt tcaactttga ctcctacctg ttgcacatga ggtgcagtcattgagcttat 49860 tctacttttc atatattcta ctcttaacat ccatttgtat atattcattgatttgaattc 49920 ctacattctt agaccatgta acagtttcat tccatttgtt ttcaatcttaaatctgcaat 49980 taaatgtatt gttgctcact gccagtcctt ttgctgaaat ttttctggtcatttgttggt 50040 ttcagtttgt cctcaagttg tttcttcaag aaatgctcat atgaataaggcctgagttat 50100 ctgtagcctt attcctgaag gactgtttgg ctagatgtgg aattgttggctcacactttt 50160 tccttgagta ccttgtaggt atttttccac tgtcttctgg cattgactgttcaatagaga 50220 agtatgatgc cagcttgatt ttatttttct tagaaggagt gtgcttttttgtgtgtgatt 50280 gctgaaagta tttcttccaa ataccaaata atttagaaat tattttattaagtgacatca 50340 gtaaaattac tagatgtttt cttagttgac cattttgggt cagttttatatccatcattt 50400 tatttacttt taaaaaattt ctatcctaca tgcttcttac tgtgcctttggtgttgtata 50460 tttttaccat gtgctcattg cattttagtc ttcatctttt taattcttaaaattcttttt 50520 ctcccatttc ttttctgagt tctgccatgc ttgtttcact acaactcctgttgactggtt 50580 agttgctcct tgagtttcta aatttctcct ctgaactttt tcttcataactgcattagga 50640 atttttcagt gtgagtaaaa agtagggttt taattttcct ctgctttgtgatgatattgt 50700 ctgttgagtt ttctttgtca gaaatgtcgc ggtgcctttt tacatttttttctatggtat 50760 tattgtatgt atacttacca cttttttgtt gcttatattg gcatgagatgagtttcccaa 50820 accatctgtt agaagggact taagggtgtt aggaacatta ggaacaccttcggagcaaga 50880 tagttttcca ggtttctgtg ctcaaggcct ctctcctcca ttgttctggtggactctttc 50940 ttcaaaatgc agccacatct cctatgcctc tcagtaactt aaggggtttaagtaatatga 51000 actaccagcc atgagttccc caggtcctga ctagttctgc taccaagggatgcacctcct 51060 accctttaaa ttgcctgcct caaatgttaa caattaaaga atttgtgacaggtatacagg 51120 agttttttgt actattctta taacttttct gtaagttcga aactacttcaaaagaagaaa 51180 ttacaaaaga gcatgcctca cttgtaaaag ggtggtcctt tctgagatctgtcacttcca 51240 aaccaccctc cacttattgt tctgccactt tctacacctc ctttccttacctccttctga 51300 gaatccccca tttaatctca gttctaaaca ttgtagttcc cgcttggtatagattctttt 51360 ctttctggga gtaaatatgt gctattcccc caccagattc ctttatacttcttgtcattc 51420 tctcatgctc agatgtggct tcttttagtc tcaagtactt tgggtcatatttacttagaa 51480 gttggcattt ttaggttttt attatcccct agtttcacta aagatatatggtattttttg 51540 tttttaacat tgtttttgtt gctctgtaca ggttctagga gaaagatgggaaaatttgga 51600 actaaattgc tgttatgttc ctactagaac ttgaagtcca gctttgaaaattattaagaa 51660 ataatttcta ggctgggtgt ggtagctcac acctgtaatc ctagcactttgggaggccaa 51720 ggtgggagga tcacttgagc cttggagttt gagaccagcc tgggcaacatagtgagaccc 51780 catctctatt aaaaaataaa gaaatgattt ctaaataacc ccttgtttaaagaaatcaca 51840 gagaaaattt tgaactgaag gacaatgaat acaatacata tcagaactggtatgatcagc 51900 taaagcaatg cttagagaga atttaaaact tgaaatgccg attttaaaagaaaaaagcat 51960 cagtgacata agcattcatt ctcaacaagt tagaaaaatc acagaaaattaaatgcaaag 52020 agaatagaaa gaaataaaag ataaaaacaa atcaatgaaa taggaaaacttctctctatc 52080 ctttagatag ggatacttca aaggttttta agaaggatag tgacatgatcatatttgttt 52140 tccagaaaga gacctctggc agcagtgtgg agaatagata gaggagaaaaaactaatctg 52200 agaagccagt taggaggctt ttcaatcact agttcaggta agagatggtgatggtctaat 52260 gtgggatgga gaggaaggat taagctgaaa aaataggttt aagaaccatgtccagaaaaa 52320 aaaaactttg ggtgtagtac tggtacatgg aacggattgg aaaaaaatagaccagagctt 52380 tactagattt ttgaaagaat tgtttttgca aataaactac aagcctggcatatcagtcaa 52440 ggttcagaac ctagagaagc agaaccagta cgaagtatag agagagagtttatgcaattg 52500 taggggctag ctaggcaagt ctgaaatttt gggggcaggc tgtcaggaagggcagggaaa 52560 ttcaggcatg ggctgaagca gttatctata agtggaattt cttgctctcagggaagcttc 52620 agccctactt ttaagacctt tcacctaatt gaatcaggcc catccacattattcaggata 52680 atctccatta cttaaagtca acagattatg gattttaatt acatctacaaaataccttca 52740 tagcaacacc tagattagtg tttgattaaa caaatggcag ctggtagcctagcaagttaa 52800 cacattaaaa acaacacctg ccatgatggc ttacacttgt aatcccagcactttgggagg 52860 ccaaagtggg aggatcactt gagattagga gtttgcgatc aggctgaacaacatagtgag 52920 acctgatctc taccaaaaaa aaaaaaaaaa aaaagaaaaa gaaaaaaattagctaggtgt 52980 ggtgcgtact tgtagtccca gctgctagag aggctgagat gggagggtggcttgagccca 53040 ggatatcaag gctgtggtga gccatgattg tgccactgca ttccagcagtgacaaagcaa 53100 ggccccatct caaaaataaa taaataaaaa gaaaaacaac cctccacacctggtctgaaa 53160 ttttctgtga ttattcagcc ctcgaaacaa ctttcaggta cagtgactgccataatgtaa 53220 actggctccc aagaaggtca tactctccaa tgtacattca gagtactgactctgagtttt 53280 ctgccaggta tgaatggtgg tactgtcaac tgaatatgga ggtagatctgctattgctgg 53340 ttagttggca gtatacacca tgtatcaatg ttaacatgtc caaagcttgaggattctatg 53400 gaacatatag atagacatat ctagtgtata tttagaaatt taggtatgaaaatctataat 53460 ggtgggagct gaaagtaaga gtgttgggac tcttcagtat gtaaatccatatttgaaact 53520 atagaagtgg aaaaactctt taggaatagt atttagattg aaacaggaaagaggaggata 53580 aagccgtcga ggaatactag catttaagaa aaaaagaact agtgagcaagtaatgattat 53640 acaaggcaat atgagacacc aaatatgatg tcaaaaaagc caagaggggaaacatatcca 53700 aaaaataaat gagctgggca caggggctca cgcctgtagt cccagctacttgggaggcta 53760 aggcaggagg atagcctaag ccccaggagt tgcaggctgc agtgagctgtaattgagcca 53820 ctgtactcca gcctgggcaa cagagtgaga cccctatctc taaaaaaagaaacaaaaagt 53880 agatgatttc acagttaaac aatgagagct ttttcaataa aatcaagagacaaggatatc 53940 agtgctatga tttttagtca ctattgtgtt tgctaacttc tgttcttcttacaggtttca 54000 gtctacatgt tacttccttg agaagcagtg tttgacaccc ttctcccccaatccagccat 54060 cccccaagtc tgagttaggt atttctcttc tgtattccca tagcacagtgtaattcccct 54120 ataatagcat gtatcacctt gaattatggg tgtttattgt tctgtctctcctgttagaac 54180 gaaagctcca tgaagggatt gtcattttat tcaccagtgt accctctatgcccagcacaa 54240 cttttggtca tattaaacaa agaatgaata aataaatcca agagcataggaggaggagca 54300 tccaaatgag cctgaaaaat tagagaaata tttgtagtag tatgtgatatcttagctgag 54360 cttaacaagt ataatgagat caagagtttg gattgaaaat gaggtactcaggaataaacg 54420 atttctgagg tctctaagat attataaaac tttattgaaa gatctaagaaaatttaatga 54480 aacacattgt ccttattaat ctataaatat agttttaatt aaaattccaatagggggcca 54540 ggtacggtgg gtatgccagt atttccagca cattgggagg ccaaggtgggaggattgctt 54600 gagcctggga gttctgacca acatggtgaa accctattgc taaaaaaagacaaaaaaatt 54660 agccaggcgt agaggtgtgt gactgtagtc ccagctactc aagaggctcagttgggagaa 54720 tcgcttgagc ccagaagtcg aggctgcagt gaactgtgat ctcaccactgtactccagcc 54780 tgggcaacag aattagaacc tgtctcaaaa aacaaataaa ataaaattccaatagggtat 54840 agtataacac ttgataagtc gatattaaga tttatatgga agaataagtgactaagaaaa 54900 accaatagaa ttttaaagaa aaataaagca gaaattgccc tacttcctataaaactgtaa 54960 cagttatacc acatggcttc aggtgcagga acaggcagat taatgaagcagagtctagaa 55020 acgagccaca tatgtgttta tggaaactgt aaatatctga tgtagtgtttcagatcagta 55080 gggaaagagt agaatattta ataaattgtg tttacacagt ttaactttaaaaaaattaga 55140 tccatcttac attagacaca aaatttattt ctaggtgaat tatatcccaaatatgaaaag 55200 caaatcttca aaattctttt aggataaaaa tgagagaatg ttcctaacaccaaacaaagt 55260 acaaagtata aactgcaaag gaaaatactg ataaacttga ccaaaatttaaaacttgtgt 55320 atcgcaaaag aaacaataaa gttaaaaaca ttagaaaatt tgtaactcatataactgaga 55380 actttacaaa atataagaac agataaattg aaaaataggt agttctcaaaataggaaatg 55440 catatagcca gtaaacacat gaaaagattc tcattcatta ataatcaagaacttgcaagt 55500 gaaatcagtg ttaccatttt ctacgcatca gatttgtaaa attaaaaagccttatgattt 55560 gcaaaaagtg caaggtgaaa agaaaaatct tatgaaatac caaatgttggtgaggatgaa 55620 gtaaaaggaa ctctcacaca ttgtttgtag tagtaccatt tggtacaactaccccggaga 55680 gcaattctca actagaagag atgaagtaaa gatgaaattg cataacttaaaacccataaa 55740 gagatatctt gcatgtgtgc ttgaaacatt atatgtaata gcagaaaattggaaacagcc 55800 taaattcctg tcagtagcag aatgaataaa cagcacgtaa atagttacataatattgtgg 55860 tgtactacat aacagttaaa taaatgaacc agatctatat gtatcaatgtgaattattaa 55920 aataatgttg tacccagaca tcatggctca tgcctataat cccagcactttgggaggcag 55980 aggccttcgg atcatatgag gccaggagtt caagaccagc ctggtccacatggtaaaacc 56040 ccatctctac gtaaaataca aacattagcc aggtgtggta gtgcacgtctataatcccag 56100 ctacttgggg gttgaggcat gagaatcact tgaacccagg aggtggaggttgcagtgagc 56160 tgagatcaca cctctgtact ccagtctagg caacagactg agactctgcctcagaaaaga 56220 aaaaagaaaa aaaaaatggg ctgggcctag tggctcagac ctgtaatcccagcactttgg 56280 gaggccaagg tgggtggatc acttgaggtc aggagttcga gaccagcctggccaacatgg 56340 cttaaccctg tctctactaa acatacagaa attaaccagg cgtgatggtgcacatctgta 56400 atcccagcta cttaggaggc tgaggcagga gaatcacttg aacccagaaggcagaagttg 56460 cagtgagctg agattgcacc actgtattcc agcctgggcg acagagtaagactccgtctc 56520 aaaaaaaaaa aaaggaaaga aaatgttggg ggtgagtaaa cagcaaaaggggctaggcac 56580 ggtggctcat gcctgtaacc ctagcacttc gggaggccaa ggcaggtggatcacttgagg 56640 tcaggagttc aagactagct tggccatcat ggtgaaaccc catctctactaaaaatataa 56700 aaatcagcca gacgtggtgg cacgcacctg tagtcccagc tactcgggaggctgaggcag 56760 gagaatcagt tgaacccagg aggcagaggt tgcagcgagc cgagatcatgccactgcgct 56820 ccagcctggg cgacagaaca agattctgtc tcagaaaaaa aaaaaaaattagcctggcat 56880 ggtggcgtgt gcctgtaatc ccagctacta gggaggctga ggcaggagaatttcttgaac 56940 ccaggagacg gaggttgcag tgagccgaga tcacgccact gcactccagcctgggcaaca 57000 gagtaagact ctgtttcaaa aaaaaagcaa aaggatatgt tattatttatataaacttag 57060 aacaaaagta tgtactaggt acatacaaat aaagtaaaat tataaaacagatgggacaaa 57120 tacacaccaa tttcaagatt ttggatcctt ttaggaagaa aaaagagaaatggaataagg 57180 tgcctgggtt gggagaagag atagaccaaa aaaatttaag aaaaatgtgagagtatgttt 57240 atgtgagcaa agtataacgt gccattggga ggcaaaaaaa taagattgattaaaaaaaaa 57300 aaaagaccta tgggccaggc gtggtagctc acacctgtaa tcccagcactttggaaggcc 57360 taggtgggca gatcacgagg tcaggagttc gagaccagcc tgaccaacatggtgaaaccc 57420 cgtctctact aaaaatacaa aaatcagctg ggcatggtgg cgcacacctgtaatcccagc 57480 tactcaggag gctaaggcag gagaatcgct tgaacccggg aggcggaggttgcagtgagc 57540 cgagatcttg ccactgtact ccagcctggg tgacagagca agactctgtctcaaagaaga 57600 aaaaaaagaa aaagacctat ggtaacttaa tgaaatggaa ataaagcctaacatttgaag 57660 ttggtttatt ttctccccag ctaatgtatt caggtatgat taacaagtaaaagttacatg 57720 tatttgaggt atacagtgtg atttttgata atacatatgc attgtgaaatgattaccaca 57780 atcaaggtaa tgaatataac aatcacctca taggtatgtg agagacataagaacatgaaa 57840 tttactctca gcgtatttca agtatacaat aataattata gtcaccatgcagtacattag 57900 gtctccagaa cttactcatc ttataactga aagtttctat gctttgaccaacatctcacc 57960 atttgcctca gccctcagcc ctgggtaact accattctaa cctctgtttctgtgagttga 58020 gctttcttag attccacatg tgagatcata cagtatttgt ctttctgtgtcttgcttatt 58080 ttatttaaca taatatcctc caggttcatc catgttgtca caaatgacaagacttccttc 58140 ttttaaaggc taaataatat tccattttat gtatatacca cattctttttatccatttgt 58200 ctgtcgatgg tcgcttaggt tgtttccata tcttggctgt tgtgaataatgctgcagtga 58260 acatgggagt gcaggggtct ccttgagata gtgattttat ttactttgcatgtagtccct 58320 gaagtgggat tgctagatca tgtagtagtt ctatttttaa ttttttgaggaacctccata 58380 ctgttttcca taaaggctgc aacaagggtt tattaattgc caataaaatgctatgaaaaa 58440 taatgagaaa tattgcactg accatcttac ttgcttacat ttcctttttgcttttgcgtg 58500 attttgattt tgcaagcctt gtacgtggta tatgcactga actaaacagcctttattgaa 58560 cttgaaatca gctggagaag actggtaatt gagcactata ataagtactgtaaaggaaaa 58620 taccaagtgc aatggaagca taaaagggag ggatccattg tagtctcactggttgaggga 58680 tgtttccctc aggaaatgac agatgggagc taggcgaaat tggtgatggaaataagcatg 58740 gaagagtact ctaagaaagg gagcaattct ctctaattgt gtgattggtcttgagaatga 58800 ttgttctatt ggcatttaat agcctaactt tcaactgtgt aattaaacttgctgtatttt 58860 accaatctaa gactcaaaat tttttgttta catttcaaca tctctgaaattgggagatat 58920 cttacaattg atggtttgtc acagttcagt tggcaggttt ctttttaaagaatacataaa 58980 atactggtgc agcttaccat caatggcatc ttagatttga tgaaatgaagaatatttctt 59040 tagcttgttt tgaggccagc ccatttgcaa cttgcatttt attattgtaattcacagatt 59100 ttaagaccat catagtggct gggcatggta gcttacacct gtaatcccagcatttttggg 59160 aggttgaggt gggtgtattg cttgagtcca ggagtttgag accagcctgggcaagatggt 59220 gaaaccctgt ctctacaaaa aatataaaaa ttagccaggc gtgatagcatgagcttgcaa 59280 tcccggccac tcaggaggtt gaggtgggag aatcacttga gccgggaggtggaggttcca 59340 gtgagccatg atctcaccac tatactccag cctgggtgac agaacaagaccctgtctcaa 59400 aaaaaaaata aaaagaccat catagtgaat ctgatgtttg ttagttatatctcccttaag 59460 atgttattta cctcttatta tgtaccagat agaatgctaa gcattttatattaattatta 59520 atactacagt gttgcatgta aattccacca cagttgtata agttggtagtatcatcaact 59580 gaagtttaga gacagaaagt cacatagttt atatttaaag cgggagaagttcaactcttc 59640 tgactccagt gcttatacct ttaacatagc tctgtactgc atcccttaagaagcaagatc 59700 cctggcaggc aaatctcaga tcttagacac attagttaaa tttatttttgtggccaggca 59760 cggtggctca cgcctgtaat cccagcactt tgggaggccg aggggggtggatcacgaggt 59820 caggagatca agaccatcct ggctaacaca gtgaaacccc gtctctactaaaaatacaaa 59880 aaattagctg ggcgtggtgg caggcacctg tagtcccagc tactcgggaggctgaggcag 59940 gagaatggcg tgaacccggg aggcggagct tgcagtgagc caagattgcaccactgcatt 60000 ccagtctggg cgacagagac tccatctcaa aaacaaaaaa ttttatttttgtttactgtt 60060 acttttcagt aaaatgtagc tgtccgtaaa acattcacta tcccattttgcttttagtaa 60120 aaagtaggca gaatatgtaa atggttgtag aatttaataa ttttatttctgcaaagtagt 60180 tagaagttca cactgctgct tttgcaagga aaacatttct agtaaataaaaatttctagt 60240 aaataaaaaa ttctagtaaa taatttatta atcacagtat tagtgttgcttacttcatgt 60300 atttgtcttg agttcaggta taagattgct aagattgaga aataatttgattttataata 60360 ttcaaattag tcctttcaga ggtagatcaa agcagaactt ttctttcgggtggaatgaga 60420 gttatcagct gattcaggca tctgggcaag aacttttagc aacatgagttcaaacaagac 60480 caactaaaga tatctggatc aggcttaccc aggcgggcta tcttcacattagaaaaacag 60540 tataaggctg ggcacggtgg ctcacgcctg taattctacc acttatgggaggccgaggtg 60600 ggcggattgc ctgagctcag gagttcaaga ccagcctggc caacacagtgaaaccctgtc 60660 tctactaaaa tacaaaaaat tagccgggca tggtggtgtg catctgtagtcccagctact 60720 cgggaggctg agacaggaga acccaggagg cagaggttgc agtgagccaagatcgcgcca 60780 ctgcacccag cttgggcgac agagtgagac tccatctcca aaaacaaaaacagtaatggc 60840 tgcaggaatc tcagtgacag tcttcatgag atctgtctct tgattctcactgtttcagtc 60900 tggttgagtt atgccctttg tgtttggtgc gcagctgtct tcttgagatcacacttcact 60960 gttgccctgg gaattccttt tgtctttgtt ctataatctt atttctttttaaaaaatttt 61020 cagaatcttc atgagaatag ggtacctggg gagcaaattt tatatatatatatatataaa 61080 atatataata tatgtatata ttagaaaatg tatttatcaa caaacttaaaatgatagttt 61140 agctctttct gtggtggaaa taatttttct tcagaaattc gaaagcatcactccattttt 61200 aaaatagctg tattgacgtt taatttacat accataaaat tcacacgttttatttcctaa 61260 gtgttatctt tcatagcatg ctattcttgt ttcacagatg cagtatcctctcttagctct 61320 ctgagcataa tttattttgg agttcttcct gtacatgctt atgcttccctcaaattcctt 61380 tttgtttgtt ttgatctctg tcttccattt tggaggcgtt catccaatatctagtattct 61440 tgattactgt ctattcagtg ttaatctaaa aacactgaat gctctgaaggcatggatagg 61500 gtttgttgac tctgagcctc atcatagggt gatttcagag ggcttaggttggggaatcta 61560 caatgttagg atccttaggt ctcttctctt ggctggtcag gttgtccaaaggtgagtctt 61620 ctaaactcct gcctaaagga tagcagtgtg gttgcctgca ttctggaagcctagttggga 61680 gttctagctg ggggaacttc tgtattcagc atttaatatg ttaaagtcatttaatctctt 61740 gtttttggta cagtcttgtg ctctcaactg tgtctggcat ccttcttccagggagcctct 61800 gttgtatctc ttccagttag tatgcctgca gatctgtgca aggatgaacaagaggcagct 61860 gttcatcaat gtgagctaga cagaggatat aggactctac ctgcttctcagactgctttt 61920 ctctttattt ttctgcatca ctcccatcct tttatcacca acttgctcacttgcttttca 61980 tctttcagaa tgttgatata tcccctgttc tgttctcctt ctaggtttgtaccttaaatt 62040 tttttttaaa ttcatagtta cagtggagtt tgagaaagga atgaaatgaagaatgataat 62100 agatgctcat tgttaatctg ccatcttaac ccagaatctg ttattaaagggcaggggctt 62160 tgtctggttt atctctaaat ttcaacccat aaaggaatct gtgacatagcatatacttaa 62220 taaatgtttg gatggttaag tcttatttta tgtctgtgtt agttatctaccactgcatat 62280 caaattattc caaaacgtag tgactcaaaa caataaacta tctcacacattttctatgag 62340 ttaggagtga cttcgatggt tctagttcat gaggctgcat tcatctcaagtccttacagg 62400 gcctgggggg attcacttcc agtaggactc acttaattgg taacaacttagttctggagg 62460 cctcagttcc ttgccatgtg gacctctcta taaggctggg taagtatcctcacaacattg 62520 tggctggctt cctccagagc aggaattcat gagagagcaa ggcagaggatataataacat 62580 taagacctag cattgaaagt tacattcttt tatttttata ataccctattggttactcag 62640 attacttaat gttcagtatg ggaagggact gtacaaagct gtgaatcccatatgtcaaga 62700 ctcattgggg accatctcaa aggctggcta ccacagtttc tgacttaagctagaaataga 62760 agagcatatt aaatccaaag taagcagaaa gtaagaaata ataaagattagagcaaaatt 62820 taattaaaaa actgtacaga atatcaatga agccaactgt tggttatttaaaaaaaataa 62880 gtaggattaa taaactccta gcaagactaa tcaggaaatt aggaaaaagcagattaccag 62940 tatcaggaat gaagtgaggg tatcactaaa aattcttcag aattgaaagattaataaggg 63000 aatggtatga acaactttat gccaattaat ttgataggtg aaacaaattccttgacaagc 63060 cacaccttta ccaaaattga ctcgaaaaag aagcctgaat agccctatatcaaagaaatt 63120 gaattcacaa ttcctcctca caaaactcca gtcccaaatg atttcactagtgagtttttt 63180 ctataatgta aggaaaaata acatcaatct ttcacaaatt ctttcagaaaatagaaaagg 63240 cttccccaac caccaccccc tgcttttttt tttttttttt tttttttttggagacagagt 63300 cttgctctgt cgcccaggct ggagtgcaat ggtgagatct ctgctcactgcaacctccac 63360 ctctcaggtt caagccattg tcctgcctca gcctcccaag tagctgggtctacaggcatg 63420 caccaccatg cccggctaat ttttgttttt agtagagaca gggtttcaccatgttggcca 63480 ggcgtgtctc aaactcctga ccttatgatc tgcccacctc aacctcccaaagtgctggga 63540 ttacatgcgt gagccactgc gcccagattt tttttttttt tttttttttttttgagtcag 63600 gttcttactc tgtcacccag gctggaatgc agtagtgtga tcatggctcactgcagcctc 63660 aacctcccga gctatgctgc tcaggctggt cttgaactcc tgggctcaagcaattctccc 63720 atctgggcct cccaaaacac tgggattata gccataagcc accatgcccggccccagttc 63780 attttttgag gctggtaaac ctggtactaa cccctgacaa agacaatataatcctctcaa 63840 caggaaaatt gacacatttt aaacattctc atgataaaaa tttacaacaaactagcaata 63900 aaaggaaatt tcttcagcta attaaaaaca cttagaaaag catacaggcatcatcatact 63960 tctgtggtga aaagattgta cacttttgcc ctaaattcaa gaacaaggcaaggatgcttg 64020 ctctcattat ttggtaatca tactggaggt cccaggcagt ggcaaagcaagaaaaggaaa 64080 taaaaggcat aatttgtaaa ggaagaaata aaactctgtt tgtagaagacataatactct 64140 acattaaaga atctaccaaa acaaacccca ttaaaattat tagaattagtacatgaattt 64200 agtgaggtca caggatgcta actgcataaa caaataatac cagcattcaacaattgaaat 64260 gaaaaaaaat ttaagtgcca ttcataacat aaaacataat ttttaggaataaatataaag 64320 atgtgcatgg cccccacgct gaaagctaca aagcattact aagaaagacaaggaaggtaa 64380 agaggtgttc ttggaccaga agtctcaaat tgttaagaat ttcattctccccaaattgat 64440 ctataaactc aatacaatag aaatcccatc aggcttactt tgggaggccaaggtgggtgg 64500 attgcctgag ctcaggagtt tgcgaccagc ctgggcaaca cggtgaaaccctatctctac 64560 taaaatacaa aaaaaattag ccgggcatgg tggcgtgtgc ctgtggtcccggctactcgg 64620 gaggctgagg caggagaagt gcttgaaccc gggaagcaga ggttgcagtgagccgaggta 64680 tcaccactgc actccagcct gggcaacaga gcaagacact gtctaaaaaaaaaaatatat 64740 atatatatat atatatatat attatataaa attgtcaagc tgattctaagatttatatgc 64800 tggtacaaag aacttaacat ttctattttg aagagccaaa aaatcttttaaagaagaacg 64860 aagttggtgg atttatacta cctgtgttca agacgtgcta taaaactactgaaatcaaaa 64920 cagtgatact ggggtaaaga ttgtcaggtt acagaatagc taccagaatagacccactaa 64980 tgtagctggt tgatttttgg caaagggccc aacataatag aatgccaaaaaggttagtct 65040 tctcagtaaa tgatgtaaca actgggtatt tgtattaaaa aaattatcctctacttaaca 65100 gcacacagaa aatttaattc agagtttatc ccaggataaa acattaaaactggtaaattt 65160 ccaagacaaa atggggaaaa tattcatgat cttggacgag gcaaagatttttttcttctt 65220 tcgagacagg atctccctat cacccggact ggagtgcagt ggcttgaacttggctcactg 65280 caacctctgc ctcccgagtc caagtgattc ccccacctca gcctcctgagtagctgggac 65340 cacaggtgca tgccaccacg cctgggtaat ttttgtagtt ttagtagagacgaggtttta 65400 ccatgttggc caggctggtc tcgaactccc tgacctcaag tgatgcacccacctaggcct 65460 cccaaagtgc tgggattaca ggcataagcc actgcacccg gctaggatttgttaataaat 65520 tgaaaaccat tgcaattaaa agcttctcct tttgagaaca ccattaagaaaatgacaagg 65580 cagccaggcg cactggctca cacctataat cccaacactt tgggaggcctaggcaagaga 65640 atcgcttgag gccaggtgtt tgagaccagc ctgggcaaca tagtaaaaaaatttttttaa 65700 ttagctgagc gtggtggtgc atgcctctat tcccagcttc ttaggaggatgaggtgagaa 65760 gattacttga gcccaggagt tagaggttgc agtaattatt atcaccactgtacttctgcc 65820 agggcaacag agcaagactc tgactcttaa aaaaaaaaaa aaatgaaaacacaagctacc 65880 gctgagaaaa cacatttgca aaacgtaaca gaacaacgct taggtccagaatatatgaaa 65940 attgtacata actcagtaag aaatagaaaa aaagtagtag taaaaatcacatacttcaca 66000 aaagaatatg tgtggtcggg cacagtggct cacacctgta atcccagcactttgggaggc 66060 cgaggcgggc agatcacctg atgtcaggag ttcgagacca gcccaaccaacatggcgaaa 66120 ccccatctct actaaaagta caaaaattag gcaggtgtgg tggtggtcacctatagtccc 66180 agctactcag gaagctgagg caggagaatt gcttgaaccc aggcagcagaggttgcagtg 66240 agccaagatt gcaccgttgc actccagcct gggcgacgag caaaactccatctcaaaaaa 66300 aaaaaaaaaa ttatatgtga atggccagtg agcccttgag aagatgcagaacatcgttgg 66360 ccgttcagga aattcaaaaa gaaccacagt gagataccac ccatacgtactagattgact 66420 aaatttaaaa gactgaaaat aacaaatgtt gacaaagatt tggagcaactagaaatttag 66480 gagtgtaaaa tggtacaagt actttgaaaa atagtttggc ggccagacgcagtggctctc 66540 gcctgtgatc tcagcacttt ggggtgccta gacaggcaga tcgcttgaggtcaggagttc 66600 aagactagct tggccaacat ggtgaaaacc tatctctact aaaaatagaaaaaattagac 66660 gggtgtggtg acatgtgcct gtaatcccag ctacttggga ggctgaggtgggagaatcgc 66720 tggaacctgg gaggtggagg ttggagtgaa ctgagatcat gccactgcactccagcctgg 66780 gtgacaaagt gagactccat ctcagcaaaa aaagaaagaa agaaagaaaaatagtttggc 66840 agtttatttt aaagttaaac ccagtcctca acttagaatg gtttgacttccgatttttca 66900 actttatgat ggtgcaaaag tagtatgtct tcagtagaaa ttgtatttatagtacaatat 66960 tctctcaaaa tgttgtgcag cagcagcaaa ccacagctcc tagtcagccatgtgatcaca 67020 agggtacagt gtactgtgtt gccagatgat tttgtccaac cgtaggctcatataagtgtt 67080 ctgagcacat ttaaggtagg ccaagctacc tattgtgttg agtagtttagatgtattaaa 67140 agcattttcg ccttaacgat attttcaaat tatgatgtgt ttattaggatgtaatcccgt 67200 tttaagtcaa ggagcatctg tattctctgg ggtttatttg agacgtagtctccatctgtt 67260 gcccaggctg gagtgcagtg gcgtgatctc ggctcaatgc aacctcctcctcctgtgttc 67320 aagtaattct cctgcctcag cctcccgagt atccaggatt atacgcgcctgccaccacgc 67380 ccagctaatt tttgtgtttt tagtagagac agggtttcac catgttggccaggctggtct 67440 cgaactcctg acctcaggtg atccacccgc cttggcctcc caaagtaagaaacagaaatt 67500 tgtttgtcaa ttataaataa atctaataaa agatatatga gttttagagtatattataaa 67560 accttagtgg aatttaaatg accttaatac attcagagat atactatgtttatgaattga 67620 aatactatcc cactgatatt ttgaacatta gtctattttg gtaaatttcttttccctagt 67680 tatagttatc tagatacatt ttcccctaaa ctcccctcct ttctgttttatttatctgaa 67740 ttctagtggc tgctcattaa ttttaattat atgagtatag tacaatttatcaatccagtt 67800 tataatacaa aatatccaaa tatgttgaca catatttagg ttgtttccattctttttact 67860 cttacaaata atgctacaat aaggatatga gcattcttat tcatgtctccttttggagac 67920 agggaatttc tctagagtat atattccact ataacagaag tgctgggttgaaaattatat 67980 acagcttcca atttattata tattccactt cctttcctaa gtgactttatcagtttatat 68040 tcctacagtc tgagagcttt cattcttcca ttggcttatt ttcagcttgttacccttaaa 68100 gttttttcta atcttgattg gtataaaatg ataggttgat attgttttagtatatatttc 68160 tctttgtctt ctatgaaacg tttgctcatg tcctttgcca ctttttattgttttattttg 68220 atttgtagga actctataca tattctagat actaagcatt tgttaaacatattgtgtata 68280 tcctctatca atctttagct ttatttcttt atttatggtg tcttttttcttatagttttc 68340 atggtttata gtttttgtat tgtgctgaag aaattcttcc ttacctttaagaccataaag 68400 atattctaca tttttcaata aatgttagtt ttccttttca tatttagttctctgaatagg 68460 aatttgttta tgtatatagt atgaggtagg ctttcaagcc tgttctgtgtgtctagcccc 68520 acagtctctt tatttgaata ggattataac aaatcttgat gaatgctagagcagacctta 68580 ttcttaagaa ttgttggctg ggcacggtgg ctcatgcctg taatcccagcactttgggag 68640 gccaaagcag gtggatcacc tgaggtcagg agtttgagac cagcctggccaacatggtga 68700 actccgtctc tactaaaaat acaaaaaaat tagctgagcg tgtaatcccagctactcggg 68760 aggctgaggc aggagaatag tttgaaccca ggagacagag gttgcagttagccgagatta 68820 cgccactgca ctccagcagc ctggccaaca gagcaagact ccatctaaaaaaaaataaag 68880 acttgttgtg gctattcttg tttcattgtg ctttcatgtt aattatagtatcttaagttc 68940 cagggggaga gaggattaca tcctgttgat attttgactg aaattgcgattgaagattaa 69000 tctggggata atttacatga atagggtgta ttataactct gtttatttgatcttctatag 69060 tatctttcaa taaaagagat acttattact tccataggtc ttatacaccttttgtttaga 69120 tttcatctca aatgccttat gttttttgtt gtgatcataa gtggtatatgtttaaattac 69180 attttaagta ggttgctagt gtataagaat ataatttatt tttatatgtagaccttaaat 69240 ctagtaacat tgctgaatta ttaattctgt aatttgtcca tagatttttattgggttttc 69300 tatgcacaca attaatgttg tgaagagtaa taattttgtt tctctctttccaacctttta 69360 actttttatt ctcctaattt gctttgctat gctagctaga acttcctataaaattaagtt 69420 gaatttacca tggagttttt tttttctttt ttatgtatct gccaggttgtaataccatag 69480 agttttggta aataactttt accagattag ggaagttccc ttctcgtcctggtacaataa 69540 gagtatttgc ttcagtgctg aattttatca aatgcttgct ttttggggactgccatctat 69600 ggaaatgatt atatgcttat ttcctgtaac atgttaatgt agtatattttattgatagat 69660 ttttaaaatg ttaaatgtaa acatttttca gtaatagcta atttgtgaaagggaaggaag 69720 gaataaacta catatatatg taggtttatt aaatattaag aattcatccaagggacagaa 69780 aagcaaacat ttgatcattg aaacatattc ctaattcaaa actatatttgctgatttcaa 69840 cccattcttt ctgagttttt tattactaaa tttaattttt acctcaaatgcccaccttcc 69900 tgtagagaag atggtcaata gtaaaatgat tcagagtgta gtggaataaacagggaaaaa 69960 tagtagaaga ggattaaaca agagaaataa ctctagcatg tttgtttagtagttacaagt 70020 aattacaact ttgtggtatc tgaaatatta gttgtgaagg ctggtgttatagcaatccag 70080 tgaataaatt ttacctgtag gattaaaaga aaggcaaaat aagcatattattcactttct 70140 atttatcttt catttacttt ttaaaaatta acccagatca atatgtgcttaagaacttaa 70200 acattataaa catataactt aaatttgata tgtatcctgt cagccttcctttttgtgcac 70260 aataatattc caacattttt cagtagaaga atataatata tatatatatttaaaactatt 70320 atggatttga gtaagatacc cagaatactg ttacaatctt aacagtctctcttttttttt 70380 taattagtta gaaactggaa atatttctaa agtttatttg gatggaagtttagaagtgaa 70440 tattgatttc tcttatcttt tccacagaaa tactcatggg ccaggcgtggtggctcacgc 70500 ctgtaatccc agcactttgg gaggccgagg cgggtggatc agttgaggccaggagtttga 70560 gaccagcctg gccaacacgg tgaaaccccg tctccactaa aaataaaaaaattagccggg 70620 tgtggtggca cgtgcctgta attccagctc ttggaaggct gaggcacgagaattgtctga 70680 acctgggagg cagaggttgc agtgagccga gatcacacca ccacactccagcctgggtga 70740 cagagggaga ctctgtctca aaaataaatg aataaataaa taaaaataaaaaataagtac 70800 tcatggatgt ataccaaaaa aataaataca tacacatacg tacacaaacacacacacgtg 70860 cacaggaaca ttcattggaa tattgtttgt aatagtaaaa aacaacccataactctccct 70920 cagaggacat gaagtacatt catacagtgg gatatcttgc agctatgagaaggaatgata 70980 tagtttcata ttagtacata caaggcacag tatagtgttt ctaatgttttaccatttgag 71040 ttctttttta ttaagagggg aagggagtac ctatttactt aattatcatgcacccacact 71100 gtaacatagt gtgcagcaat taaagggtgt actgacatga aaagatctctaagatgtatt 71160 gttaattttt aaaatcatga taaaatgagt catagaacag ttatctgatttcatctaaaa 71220 agttattcct acatttttat ctttgtaaat tagtattttt gtttttttaatcacctttct 71280 gacaaatcta agtgcatatt aaaaggagag gatgtataca tacctaagtactaacaatgg 71340 ttacttatta gagctatttg gagcgaggag gttcagactg gagagggtctttactttgta 71400 tatctctgta ttgtttgagt ttttatgatg aaactttatt catgtattatttgtgtaata 71460 aagaagtaaa aaacaatatg cactcagtac caaaaaatga agttataaagataaaaatgt 71520 tttctcgtag taaaaaatat gttaaaaaaa attttttttt gagacgaaatgtcttgctct 71580 gtcacccagg ctggagtgta gtggcgcaat ctcagctcac tgcaaccttcgcctcatggg 71640 ttcaggcgat tctctggcct tagccttctg agtagctggg attacaggcatgcgccacca 71700 cacacagcta atttttgtat tttttagtag agatggggtt tcaccatgttggccaggctg 71760 atcttgaact aatttcaagt gatcaatgca ccttgggctc ccaaactgctgggattacac 71820 ttgtgagcca ctgtacccag ccaagataat tttttttttt ttttttttttttgagacaga 71880 atctcactct gtcacccagg gtggagtgca gtggcgtgat ctcggctcactgcaacctcg 71940 gcctcccagg ttcaagcgat tctcatgcct cagcctcctg agtagctgggattacaggca 72000 catgccacca cgcctggcta atttttttgt attttcagta gagatggggtttcaccatgt 72060 tgcccaggct ggttttgaac tcctgagctc aggcagtcca cccgcctcggcctcccaaag 72120 tgctgggatt acaggtgtga gccaccgtgc ccggccaaaa atattttgaagaaaataaaa 72180 tacccacagt agcctcacca cccacattta tccagagata actactgttaaagctttgaa 72240 aatatcctcc cagaggtaag gaacatttta tggaggacat ttaatgctaaaccaaggaaa 72300 gcaagagaaa gatccccaca taaaaagttt acgctcctga cctcgtgatcctcccgcctc 72360 ggcctcccaa agtgctggga ttacaggcat gagccatcca gcctggccaacgtggtaaaa 72420 ccttgtctct actaaaaata caaaaattag ccgggtgtgg tggtgggcacctgtagtccc 72480 agctactcag gaggctgagg caggagaatt gcttgaacct gggagggggaggttgcagtg 72540 agccgagatc gcgccattgc actccagcct ggacgacaga gtgagactccgtctaaaaaa 72600 taaataaata gataaataga taaataaata aataaagttt acacttagctgaacagacag 72660 tgggcatttt tttttttttg gagtagaaaa atgacatgat aacatgcaacttactgtctt 72720 atggttttac tttttgttgt atccccagtg cctatataac acataatagccattaataag 72780 tatgtgtctg agaaatgagt gatggtgatc tgctaaccat tggtgaaactcttctagttc 72840 caaaacccat ggttaagaaa tcatagtggt tgactgttaa aaaaccacccactgctcaat 72900 aatattggac agataaccct aaatattact atctgtagac tgagcacagtggctcatgcc 72960 tgtaatccca gcactttggg aggctgaggc aggcggatca cctgaggtcaggagttcaag 73020 accagcctga ccaacacgga gaaaccccgt ctctactaga agtgctatattggtcggacg 73080 tggtggtgca tgcctgtagt cccagctact tgagaggctg aggcaggagaatcgcttgaa 73140 cccgggaggc ggagattaca gtgagccaag attgtgccat tgcactctagactgggaaac 73200 aagagtgaaa gtctgtctca aaaaaaaaaa aaaaatatat atatatatatatatatatat 73260 atatgtatat ttgtagctta gatttagcac caatgactaa taccactgttttttcaaatc 73320 attacaagac agggaaaact ctctttagaa tagatagtct agggtggcatagaaagcctt 73380 gtttaagaaa taatactggc caggcatggt ggctaacacc tataatcctaccactttggg 73440 aggctgaggc gggaggattg cttgagctca ggagttggaa accagcctgagcaatatagt 73500 gagaccttgt ttctattaaa aaaagaaaaa aagaaataat acctaaccatctcatctgcc 73560 acggggctcc agccttcttt gtctctttca agccttgtct cacccagaacacttctctat 73620 agagaatgga atgaatcagt tacttacgat gaaagagata gattataaaaactgtagtat 73680 ttggccattt tgtatttgtg cctcagaggt caaattaaaa ctgcttaattttggaaattt 73740 gtatgtacat aaatgtgcat aaaggatcat tttaacccta tagatagttatcctggagcc 73800 taagatgaag tttctgaatt ttttagggaa acattaattt gtattgtttgtgtttttctt 73860 cttagtttct gccactgcct tctacaaagc acaacctgta attcagttcatgtgtgaagt 73920 tcttgatatt cataatattg atgagcaacc aagacctctg actgattctcatcgggtaaa 73980 attcaccaaa gagataaaag gtgaattaat tagcatttag cacaacttaactataaaatg 74040 catggatata acaacctttt attaaaatgt tttcttatat aattaaaatctatgtaacat 74100 aaccagtaaa actatacatc aattataaaa taaaatatta cattaaattcattactataa 74160 catcttaggt ggtatttgaa agaaatgcca agtaatggag ttagtatcactaccagctaa 74220 ttttttgtta atactttccc ttactagctg aagggataag aatgtctcatgggaaaatgc 74280 tttagtaata ctgttaaaag taggaagctc actccaaaaa tatttgttatattttataac 74340 tagaattata gaactagaac atattctaat ataatgactt ccttatttttctgaaatgat 74400 agttatttat tttagtcctt taaaaacgta taaatgtggg gctagcctgtttcatgtgtt 74460 gttctagaat tatagttatt taatataagt tatagagttg actgttgatcttatctatta 74520 tacttagtaa ctaaaatttg tacagtgctg ttgcatcata tgtacatttaactcagataa 74580 attcatctct atgtggctgc ccaaaaataa aaataaaaat aattttaatagtgttctgaa 74640 atctgtattt tatatgtatt tagtctgata ctccctaatc agaatgaataggataagttc 74700 tatttatatt gttagatctt aaatagccag aaataattat gttgtttttaagattttacc 74760 tatttattgc agataatttt cagcttgtgg tttgaatgaa gcatttaagtttgttcactg 74820 aacatctttc agttccccca aatcaattag agtagtttaa tacattctgcttgctccagt 74880 aataattcat tcttcgccat tcagtaacca agagagagat agtcattgtaataaaatatt 74940 aaaaggaaga tccaccctgc tttgaaaata agcatccatt tatctcaagttatttaaatg 75000 ccaaagtaag aaactgttta tgtaactgtt ttgcaaccat accattgtcctttcatgatc 75060 tgtattaact gcacagctag cattgtcagg aggttagtgc attacctaccattaatatgt 75120 gatcatctaa atgccatggt ttccagtgga gttgctgctt agttaccacctctgatatca 75180 tgaagtcact tacattattt tgcggtaact atgcaatcaa ctcatatcaccactctatca 75240 cagaaaatac aagatgtaca gaacaaggat tcaactgctg cccgaagagcatggactcga 75300 tcttaacttc aactgctcag gggcccaaag aaatgactga aaaaatgactagaaagcata 75360 ataaagttga tgttatagtg aaggtaaagc caagtttata ggttaaatatttattaaagc 75420 caaaagtgtt tttttggttg gatggagggt ttggggaggg acctgggcagagaattatct 75480 agtccacttt ttaaaaattt caactttttg ctagcaccta gattttggtctaattcttag 75540 gtatttatga tcctagtttg ggggtggaaa aatctgggtg ggatccaattgacaaactga 75600 ttattttttt cttcattcca agtaaaatag ggcttttttt ttttttaaagaaggctcttt 75660 aaagtacatc atgtattcct actacagtaa cccaagagaa taagagttttgtgttctctc 75720 ctgtatctat gggtttttta gctggtatga agaatttgat gctagctgttaaataatgac 75780 acaaagtgtg caataaagaa tagattttct tagtatattc atctttttccagttgaactt 75840 gtacctaaat atttaataat aactttggag tggtttcttt atccagtaactcgtgatagt 75900 taaacagcag aaatactggg aaaatgtata agtaccatat atatatatctatatatgtgc 75960 caacttattt ttagtaaaag ctttataggc ttggagaacc ttattaatatttttttaatt 76020 caaggggaag agttgaaaat aaattgctaa gtggtgcaaa taacagaaatttgttttaga 76080 cttttctatt gtgttctccc ctgaacataa tatttttacg tcttgtttcttcatattatt 76140 agtatttcct tcttttaaga tcctccttcg atagtatttt attttctagtctcctttata 76200 atataaagac atagtaattc tctagttgat ctgacatttt tgtcttataaaggtagttgt 76260 aatgaaaaat tactactcac tgattcaagg aggagcactt ccaaagaaaaacaaacccat 76320 accaatctgg tacagggatg gataccgtgt ccatttaaca tcttaatttgttctgatcta 76380 tcagaagaaa atatcattta tatgattgag ctttgtgttt ggtactaattttcttaccta 76440 attgcaggta gcagttaggt aagaaaataa attttaccat agcaggcattctcaacctta 76500 gcagcattga cattttgggc tggataattc tttgttggga taggggtagggctgttctct 76560 gcattacagg atatttcata ctatccctgg cctctactca atagaaagcagtagcatctc 76620 tagtcatgcc tatcagtttg tctccagaca ttgccaaatg tcccctgggggacagattgc 76680 cctctggcta agaaccactg cagtatagga atcacaaata tgaaagagaaatatgacaaa 76740 gagtaataac cataagtatt atcagggaac taaaagttgt tctccatctaccccattttg 76800 gactttatcc cttggagtct aggccaaact ttagaaagga gttgcttatctgtgacctct 76860 gtgtacctgt taactcttct ctaggctaag actgcactaa ccacatgtagctacttaaat 76920 ttaaattaat taaaataaag gccgggcgtg gtggctcacg cctgtaatctcaacactttg 76980 ggaggccaag gtgggtggtt cacctgaggt ctggagttca agaccagcctggccaacatg 77040 gcgaaacccc atctctacta aaaatataaa aattaactgg gcatagtggcgggtactgta 77100 atcccagcta cttgggaggc tgaggcagga gaatcacatt aaccctggagacagaggttg 77160 cagtgagcca agattgcatc attgcactct gacctgggca acaagagcaaaaaactaaaa 77220 aaaaaaaacc taaataaaat taaattttca gtttcttagg tacaccagctacatttctta 77280 tgcttagtaa catgttatta gtgattacca tattagcata aatgtagaacattccatcac 77340 ctcaagaaaa ttctctttga cagcactgct tcagaaaatt catctcgaagaacttcagcc 77400 aacagcttaa agcccatttc ttcacattct agagcaggcc tgggatccttgaacccctag 77460 agtccacaga tggacggcag ggatccaaga actcctaaaa tcatgtgctaaattatgtat 77520 atgtgcattt tgctgagagt ttattgctta catcagattc tcaaagggatcttgactcca 77580 taagagggta gggatcacct actttaggaa ttattttggt tacatttgatttttaatagt 77640 attggaaaag agctgtgtca ctatattata cctctataaa agtgtcactttgcttctgtt 77700 aaaaatgcct ggaatttttt tctcctgctt tataaatctt taaaggacttctttcttgag 77760 ctttacaaaa ttctgatctt caagcagtat gtgatgactg ggctaggtaaatatcacata 77820 atttgaaatt atggaatacc ttgagaatta ttggatctcc tctttcattcctccttccct 77880 ctcccagcat aaatacctga atttattata aacaggattt tttattaatggaagattttg 77940 gcccttgatc tatgggcttt gcaaatttta tgattttatt ttttaatgtgtagagcttga 78000 tggggtaagg aaaaattttg ccattaggtc tgtcatgact gtgaccattattagcaatgt 78060 tatatgtaaa atctggtgtt tatatcatct tgcctgtatc acagaattttttgtctgttc 78120 agaattgagt ttttatggta atgaacaggc tttataagta taaatattttacatgtgaca 78180 gttctgtaac ctccattttt cttgttggga acaggtttga aggttgaagtgactcattgt 78240 ggaacaatga gacggaaata ccgtgtttgt aatgtaacaa ggaggcctgccagtcatcaa 78300 acgtaagaaa agtttgtcag agcagcgatg gtgtgaggca gcttgctctagttagtgggg 78360 ttgggagttt ttctggctca taatgggcaa gaattgttca tgtgtacttttttttcctca 78420 gctttccttt acagttagaa aacggccaaa ctgtggagag aacagtagcgcagtatttca 78480 gagaaaagta tactcttcag ctgaagtacc cgcaccttcc ctgtctgcaagtcgggcagg 78540 aacagaaaca cacctacctg ccactagaag taatgccttc acactgctaattaataccct 78600 gttgttcatg attcttttgg ggtcttttat ggccgataac ttacctcatacagaacattt 78660 attttggaat atgaactgtt tttaagtttt aatttattct aaattttctgtaatgaaata 78720 acacgaaaca agttcatcca taatcctacc actctaatgt aaccactgttaacattttgg 78780 tatactatac ttctttcagt cttcatctgg tgtacatttt acttaattatacttaattac 78840 tgtagagctg tcttgtttac tactttttta cttaacatat tgaaaataagtggtttaatg 78900 aagttgattt taaattttta tttacatgta attggcacta agtaagagctaaaaaaaaaa 78960 gatatataca tgtagtacaa aacttttttc catttgtagg tctgtaatattgtggcaggg 79020 caacgatgta tcaagaagct aacagacaat cagacttcca ctatgatcaaggcaacagca 79080 agatctgcac cagatagaca agaggaaatt agcagattgg ttagtacttaaccttagaaa 79140 tgagaattta aaacatatta gggtgaactg taatactaga gaaccatgtccttatcaacc 79200 cataccttat gaccatttca tggactgtca gaattaaaag caatcatggaagtaatctaa 79260 tgttcttgat acaagccttg gtgggctata tgagaagggt taggtccttctcatttaacc 79320 ctgagctttt taagtagatc cagggaacag atcctctcag aagaaatgtttccatttagt 79380 aaactggaac ctccacctaa aagaggtggg aaataggagg aaaagtcaaagaattatgac 79440 tagcaatact aaaattcttt tttttttttt tttttttttt tgagacggagtcttgctttg 79500 tcacccaggc tggagtgcag tggcacgatc ttggctcact gcaagctctgcctcccaggt 79560 tcacaccatt ctcctgcttc agcctcccca gcagctggga ctacaggcgcatgccaccac 79620 gcctggctaa tttttttgta tttttagtag agacaggatt ttaccatgttagccaggatg 79680 gtctccatct cctgactttg tgatccgccc gcctcggcct cccaaagtgctaggattaca 79740 ggcgtgagcc accgcacccg gccaatacta aaattcttaa cactcagtctaagattgctt 79800 agtccccagt atcacagtgg cagctgtaca gtctgaataa agaaatggctgggctcagtg 79860 gctcacatct gtattcccag cagtttggga ggccaaggcg ggtggatcacttgaggccag 79920 gaatttggga ccagcctggg aaacatggca aaatgccatc tctactaaaaacgcaaaaat 79980 cagtggggca tggtggcatg tgtctgtaat cccagctagt caggtggctgaggcatgaga 80040 ataacttgaa cctgggaggc agaggctgca gtgagccgat cacaccactgcactccagcc 80100 tggacaacag agtgagactc tgtctcaaaa aaaaaaaaaa agaaaaagaaggtcgggtgc 80160 ggtggctcat gcctgtaatc ccagcacttt cggaggccga ggggggcggatcacctgagg 80220 ttgggagttc aagaccagcc taccaacata gagaaactcc gtgtctactaaaaatacaaa 80280 attagccagg catagtggcg catgcctata atcccagcta ctcaggaggctgaggcagga 80340 gaatcgcttg aacccgggag gcgcaggttg cggtgagtcg ggattgtgccattgcactcc 80400 agcctgggca acaagagcga aactctgtca aaaaaaaaaa aaagagagagagagagggag 80460 ggagggaggg aaagaaagag aaaaagagag agaaagaaaa gaaagaaaggaaatagaaga 80520 tagctttaac cacgtaaggt ctctgtgctg tctcattgtt ctaaatgaggggaaggaagg 80580 cgttaccaat accgctttat aaaactcata gagtaaggca aaactcaagattaaagggca 80640 tctgaatctt atctggctct gttctcgaat accattttaa ctgcccaagcaaagagaaaa 80700 aaatctggaa atatgaatat ttcccaaaac aacttaataa tacttccaaaagcaatttta 80760 aagattattg aaaataggga tgcaaaactg gctcttttaa tttattttttaaccttagta 80820 atttcacctt tggcaaaaac tggctctttt atttggtaag ccaactgcagaaaaaatact 80880 tttttttttc cttattgact cttctgtaaa gagtcaaaga aagttattacatgatattta 80940 attaggaaaa tatatgctgg ttttcatctt ggatttttct ctgattctactataagattt 81000 taaaaactct caaaatcaga gactaagacc tcttactttg tctataaaagtctcaataac 81060 tcactgttct aattcattta ttacagctta actgttcaga aagtatcatatggtagttta 81120 aaaaaaataa aacagacttt ggagtctaac aaccctgggt tggaagtctggcttctccac 81180 ttacttactg ttactgttac tattattata tttatagtat ctggaaagataatgcagtga 81240 ttaagagtag acactctaga atcagaatat acatggtttc acattctgtctacatcactt 81300 acttagctgt gcggtttttg gcaatcattt tactttactg agctttagttgtatcctctg 81360 taaaatacag attataaaag aactgttttt gtagctataa aatacttgtgaaaatgtatc 81420 attaagcact tagcaccaag tacatcatgc agcaagtcat agtatattagttgttatagt 81480 atagtcgtca ttattcgctg taaccttatt acaggataca ctgaagtacagaaatgcatg 81540 gcaaataaga ttgaaagaga tgccccattg gtgcctaatt tagaaaatggggcttacacc 81600 tgtaacccca tcactttggg aggccgaggc aggactatca gttgagcccaggagttcaag 81660 accagcctag gcaacatagt gagaccccat ctctacaaaa aaaatgttaattagccaggt 81720 atgtggcatg cacctatagt cccagctact caggaggctg gggtgggaggatcgcttggg 81780 ccccaggaga ttgaagttgc agtgagctgt gattgcacca ctggatgacagagcgagacc 81840 ctgtctcaaa aaaaaaaaaa agaaaagaaa aggaaaaaga aaagaaaatgacttttcctg 81900 cttattgtgg agagattcta tatagcagca tgctttaagc atgaatttcagtgagattca 81960 acactgaatt acatgaagtt ctttgattat atgtatggga atatataaaaattataacct 82020 gtgactcata ctggctttct tatctgtctt taagtgtatt ccattaaataaattttataa 82080 tatggcactg aaaatagtca agtaaatttt atataaatta taaatcaaataatttttaaa 82140 agctttaaga tttttaatat ttcctatcaa actctacaca aaataaattttaatagaatg 82200 tatccagatc tcccatagtc agaggttctt aacctctttt taggtgagatatttctttct 82260 ccatccaaaa gcatattagg ttcaatcata caaaattaac attttcagctgggtagtaca 82320 tagtggtgca tgcctatgtt cccagctatc taggaggctg agagaggaagatggcttgag 82380 cccaggagtt caaggccagc ctgaataaca tagcaagact ccatctctttaaaaaaaaaa 82440 aagttttatt atcattttca tggatttttt ttttcttttg agacagggtcttgctctgac 82500 ccccaggctg gagtacagtg gtgcaatcac agcccactgc agccttgagctcccaggctc 82560 aagcgattct ctcacctcag cctcccaaat agctgggacc acaggcacacagcaccacac 82620 ctggctaatt ctttttaatt ttttgtagag atgaggtctt gctttgttgctcaggttggt 82680 cttgagctcc tgagctcaag cagttctccc acctcagcct cccaaagtgctggaattaca 82740 gatgtgagcc accacgccca gcctaatttt ttaatttttt tgtagagacagggtctcact 82800 atgttgccca ggctagtttc aagcttctgg gcttgagcaa tcctccagcttggcctccca 82860 aaatgctgag atcacaggca tgaactacca tgccaggcca aaatgtattattcaacctaa 82920 tgcatacata caaattttgt ataaattacc aggaggtcac agaatttctgcagtccattc 82980 atgacccatg gtccccaggt taagattcgc tgttctgtag ttagagcaccattgattaga 83040 cctgttggaa aatatattat ggacacagtg aagaaaaaaa agagggttctaatgcattaa 83100 agtaggggat ttgggggaat tttgagagta actacaaact ttttctatttttaggtaaga 83160 agtgcaaatt atgaaacaga tccatttgtt caggagtttc aatttaaagttcgggatgaa 83220 atggctcatg taactggacg cgtacttcca gcacctatgc tccagtatggaggacgggta 83280 aagtctcttg ttaatgtttt aatcatacac atattgtctg taagtatgaagagaaaggca 83340 tatcagaaat atttcaattc agcgatttga aatgtttact ttctgtttattgaaaatttt 83400 tgttcttttt caccatgtta tttttttctc ctcgtgtaga atcggacagtagcaacaccg 83460 agccatggag tatgggacat gcgagggaaa caattccaca caggagttgaaatcaaaatg 83520 tgggctatcg cttgttttgc cacacagagg cagtgcagag aagaaatattgaagtaagac 83580 atgtcattac cttggctttg ggactttttt gtgtttagac tttaaattactcatctaatg 83640 ttctaacaga tgttgcctta atatgaagta tatgtaatca ctgaaccatttttttttttt 83700 tgagacagag tctcactctg tcacccaggc tgaagtgtag tggcgtgatcccagctcact 83760 gcaacctcca cctcccaggc tcaagcaatt ctcctgcctc agcctcccaagtagctggga 83820 ttacaggtgt gtgccaccac gcccagctaa ttttgtattt ttagtagagatggggtttca 83880 ccatgttggc caggctggtc tcaaactcct gacctcaggt gatctgcccgccttggcccc 83940 ccaaggtgct gggattacag gcatgagcca ctgtgcccgg cctagtcactgaacctttaa 84000 aaatgtttct tcttgaccag gcacagtggc tcacacctgt aatcccagcactttgggagg 84060 ccagggcggg tggatcacaa ggtcaggaga tcgagaccat cctggctaacatggtgaaac 84120 cccgtctgta ctaaaaatac aaaaaattag ccaggcgtgg tggtgggcgcctgtagtctc 84180 agttactcgg gaggctgaag caagagaatg gcatgaaccc aggaggcggagcttgtagtg 84240 agccgagatt gtgccactgc actccagcct gggcaacaga gtgagactctgtctccaaaa 84300 aaaaaaaaaa aaaaacttct caatagttcg gtgaaaaaat ttgttagtcatagtaaattc 84360 cttgattcat tctattagac atccttatag actactgtga atggaaaaaacacaagctca 84420 aaccatgttt tttctctgct ctcacaccag agcaatcaac acagaacactcttgtgacta 84480 aatgtgggga gggtttcccc acacaccaag caaacaatca gttctgcagtggacaccagc 84540 tgggtgtcct ccaattcagt tattacacta tcttcctgga aatagcatcagattccacag 84600 attcagggtt cactcccaca aggctgcact ctgcttcaga tgccagtagcaagtccaggc 84660 ctctggtcat gggttcccat gaccccctct tcaagataga ttcgtctgatagagcagctc 84720 acagaactag ggaaacactt actatgttta ctgttttatt ataaaagatattccaaagga 84780 tgcagtaaag atatccatag ggtgaggtat ggggacggag tgtggagtttacatgccatc 84840 cctgggtaca ccaccctcca ggaacctttg tgtgttcagc tggccagaagctctccaaac 84900 cccatccttt tggattttta tggatgcttc attacatagg cctcattgattaaactgtta 84960 gccattagtt atcaacttaa ccttcggccc ctttcgcctc cttggaggttggggatgtgg 85020 ggctgaaagt cccagccctc taatcctgcc ttgcctttct ggtgaccagaccccatcctg 85080 aagctaccta taggctacca gccgttaggc aatcagtatc atataaaaaagataacactt 85140 aggagtttct aaggatttta ggagttgtct gctaggaaat gggaatggccaacaaatgca 85200 tatttcacaa tatcacaact actttttgtg ggatagtagc agcaagcatcattgtctttc 85260 gatgacagtt taggtataat tcccagattt gccactacct ggatttattaccatggataa 85320 tttatttaat ctcattattc ttcaatgttc acctttgtaa aatagagctaatcatatctt 85380 ctgttaaaat aaaaacttca gacaaattaa atttaacaga gtttataattgagcaaagaa 85440 tgatttgcaa atcaggcagc tcctggaaca agaacaggtt cagagagacttccagcagcc 85500 gcatggttga agaagattta tggacagaaa aaggaaagtg gcatacagaatatggaaatg 85560 aagtacagaa acagatggat tggttacagc ttggcatttg ccttatttgaagatgatttg 85620 aacagttagc tgtaattgat tggctaaaac tcagtgtttg gtacacaagtagcttatcca 85680 ctatttaccc atccagttag gttacagttt tttgtttttg ttttgagacagagtcttgct 85740 ctgtcaccca ggctggagtg cagtggcgtg atctcggctc actgcaacctccgcctctca 85800 ggttcaagcg gttctcctgc ctcagcctct agagtatctg ggactacaggcctgcactac 85860 tatgcccggc taatttttgt atttttagta gagatggggt ttcactctgttggccaggct 85920 ggtctcaaac tcctgacctg aggtgatctg cctgcctcag cttcccaaagtgctgggatt 85980 acaggcgtga gccactgcgc ctggcctagg ttacagttta cgatgttcagagaaacctct 86040 agactgaact taaaagatgt aaggaggtag ctttaggcaa aacttaatttaacagttatc 86100 ccctttgatc tatcccccca attttttttt ttaatggatt ggcattgatgtcagtcacca 86160 tcataaactt acttatttgg tctcaaatcc cactgggaaa tagcagaacaatgggttttg 86220 taaaatggga acaaggactt caggttactt tttcctaagg gtaagagtagaggggacctc 86280 cttgtgctga aatttcctgt tttcaggaga aaaacaaaac ctggtctattttaggatcta 86340 tctctttctt taatgtttca gtgtaattgt ctcacgctta gcatgagtgactccattttg 86400 gtttggtatg gtctgttggg gcctcatgca tgagcttagt ccaaaacaatggcctcccac 86460 aattttgttt aaaaattcct cccttttggt taggtcctca cttaggtaagagtgtgacca 86520 aaacttagga ccttagcacc actctgttac caagattttg ggtttctggtctcagtacgt 86580 catttataag tatggtgttc ctcatgctca tacatttctt tgagtttctgttccaattca 86640 agagagacca tttgacatcc tacagatggc catatgcaaa cacttaaaacttttgagaga 86700 atatagtatc ccagggagac tactattatg actctcagga gaataaccctaagagtttgg 86760 agtatgctcc ttagccaagg tccccatgaa ccaagccacc taaaatcaaatagatcgaag 86820 aataagctag aataagagtc tacttgtttc aaccaagcac cctgtttgttaatcccctac 86880 gactgaatct gttaatatcc aatgtattcc tccatgttca ataagaagtagcagcagctg 86940 cacagatact tctgtttagc cagtaagtaa tctagagcaa ttctattatctagcacaact 87000 ttagcaagat aatttaaagt ctcttgtata accatagtct ttgcagtagaatctgctaaa 87060 gagcctataa atttctaata ggctagaaaa tttagagaga taaatttctaatcattgcct 87120 cattacatta actccaaacc ataggccagg cgcagtggct cacacctgtaatctgcaatc 87180 actttgggag gctgaggtgg gcagattgct tgagctcagg agttcaagaccagcctaggc 87240 aacatggcaa aaccctgtct ctactgaaaa ttcaaaaata atccgagcacagtggtgcac 87300 gcctgtggtc ccagctactc aggaggctga agtgggagga ttgcttgagcccaggaggca 87360 gaggttgcag tgagccgaga tcacgccact gcatcccagc ctgggcagcagagccagacc 87420 ctgtctccaa aaaggaaaaa acacaccata gaaaaataaa ctaacaaataatgcccatcc 87480 agaagagtga aggcctcctg gcactattct ctttaacctg tattgacaatgttctctttt 87540 tttttttgag gctggagtgc agtggcatga tctcggctca ctgcagcctctgcctcccgg 87600 gatcaaacaa ttgtcctgcc tcagcctccc aagtagaaca ggcacgtaccaccacgccca 87660 actaattttt tgtattctta gtagagcggg gtttcaccac gttagccaggatggtctcca 87720 tctcttgacc tcgtgatctg cccacctcgg cctcccaaac tgctgagattacagttgtga 87780 gccaccatgc ctggccgaca attttctctt taaattatga tgtaggttaagaggagttga 87840 ccagtgctct gtttctgact gattatgaag caaaaaaggt accattaaaatttctcaccc 87900 acattggccc ttcatcttcc ctctatcaag gcgtaaactt ctctgtgtgtgagggttttt 87960 gttttttttt gttttttggt ttttgttttg agacggagtc ttgctctgtcaccaggctgt 88020 gatcttggct cactgcaacc tccatctccc gggttcaagc aattcccctgcctcagcctc 88080 ttgagtagct gggactacag gcatgtgcca ccatgcctgg ctaattttttgtattttagt 88140 agagatgggg tttcaccatg ttggccagga tggtctcgat ctcctgacctcgtgattcac 88200 ccgccttggc ctcccaaagt gctgggatta caggcatgag ccaccacacctggccaatat 88260 gaggtttttt ttatccttca caaataaaag tataccttgt gagtgtacacaagagacccc 88320 tttttcagtt tagttgttca taacaggcat gaacttggaa aaaattgagagccaaaagcc 88380 tcatgatagc agagaagtct tgatccacaa tcttgggaaa tctgtccacatctaggaggc 88440 catctgcttc tcgggagaaa cttacctcat tagctttacc ttaagttctccctctgatgg 88500 gtgtgtggtt ccaagagtct gggtgggcct ttctaagttg tgagattacaaacccaagct 88560 tcagggtcct gaagtttcgc tgcagtgtgg gtgacaaggg gagtctttctctgatgtgtt 88620 tccaaaagat ccagcctctg aattctatat catgaagggt ttgcttgtcctgagtcagtg 88680 gtccatgaaa agctttcttt acctggtgaa aatacacttt ggcataatacattacagcct 88740 tgcagcattt agtcacgtta aggtttagga gcataagata cagaaggttctgttattagg 88800 agcataagcc ttccagtgac tatttcataa gggttcaact tttgttttcccatggaagtg 88860 gatctgtttg tcatcaatct gaaacatctt tgaccaaggc aatccagattattcagttag 88920 ttttgcctaa tgctcttata tctgtaatac cttatttaac tgttttacagccagtccagt 88980 gaggcaagta tctctatcac tggagatttc ttcagcaatg ttctatgagagaaacacatt 89040 tcctaataac cttttagctg ctgttatagc atcagcccac ttgtatgagaaagctcctgt 89100 acaaccagaa aatatgcact gaaaatcaca attgaatgaa atccctctgtaaagtgttca 89160 gatgtagcag aaaggtacct gaagttttgg ttgtcttctc aagattatgggtttgacaaa 89220 ctatacattg gtcataaacc attttagcaa tttagaacag tcacaacaccaatatatatg 89280 taaggtgttt gtttgtttgt ttgtttgttt tgagacatag tctcactctgtcacccaggc 89340 tagagtgcag tggtgcgatc tcggctcact gcaacttcca cctcccgggttcaagcaatt 89400 cttgtgcctc agcctcccaa gtagctggga ttgcaagtac ctgccaccacacccagctaa 89460 ttttttgtat tttttaagta gagacaggct ttcaccgtgt tggcccaggtggtctaaaac 89520 tcctgacctc aggtgatcag cccaccttgg cctcccaaag tgctgggattacaggtgtga 89580 gccaccatgc ccggccagta tatacatttt atctcttcct tgatgaatcatggaatacag 89640 cttctagtaa tggaattttt aaggactcag gaaggagcag gcagccggctgtccagtctc 89700 tctccatgag tccatgctta acactggaat tgtatcctct tacatagcaattttctttct 89760 ccaatggagg tgcacagcac tgtttattag atgggttatc ataggtagtttgacctggac 89820 catggagttc attcaaatta tgtatcttaa tagtttcagt actgactgagttagcatgaa 89880 aatctggcca agtattttct tggtattcat ttaattcttg tgctgcttgagttagcagtt 89940 ttatatatca ctctgtctct tcaatatggt tctggtaatt cttactcagtccaaacgata 90000 tgatcctaaa gttaccagaa acctatcttc aggagtgctt accaaggtccatttcatctt 90060 ttccattaac ctccttgaag acaaaatagg attttatttg cttgtgaagttatttttaat 90120 aactgccata catttattta tttatttatt tatttattga gacggaatctcgctctgtca 90180 tccaagctgg agtgcagtgg tgctatctcg gctcactgca acctcctccttcctggttga 90240 aactattctc ctgcctcaga ttcccgagta gctgggacta caggcgcatgccaccatgcc 90300 ttgctaattt ttttgtattt ttagtagaga tgggtttcac cttgttggccagactagtct 90360 cgaactcctg acctcaagtg gtctaccaac cttggcctcc caaagtgctgggattacagg 90420 ggtgagccac ttgcacccag cctgccatta ctttatttat ttatttatttatttagagac 90480 ggagtctcgt tctgtcaccc aggctggaat gcagtggcac aatctcggctcattacaacc 90540 tctgcctccc agggtcaagc agttctcctg cctcagcctc tcgagtagctggtattacag 90600 gtgtgtgcca ccatgcctgg ctaatttttt gtatttttag ttgagatggggtttcatcat 90660 gttggccagg ctggtctcga actcctgacc ttgtgatctg cccacctcagcctcccaaaa 90720 tgctaggatt acaggcgtga accactgcgc ccagccgcca ttactttgaacagcagaaac 90780 cgcaattact tttgcaccaa cctaatatta gaaactgctt tagaattaagtaattaactg 90840 tggaaatgac tttaaatggt cataaagaca caattgagaa ggaaatttggttatttctgt 90900 ggcctacaat agtttaacgt aataaccata attatgtctg ataacatatactgagataca 90960 tgagaatttt cataatctta tacaattttg gaatatatat taatatttataaaaatataa 91020 ctcgatggag tttaaacatc acttcttatt tgacactgtt tctcatgtaattttacgtat 91080 aaaataagcc tgtttattat ctcttttgac tgttgtaggg gacctctgtaacatcccaaa 91140 gttaatttga ggtcaaaaaa agacttaatt ttgaatttga aatttgatttggggaagctt 91200 gtccaatatg tcaaagattg aaaacacttg gcccaaatag gatcacaggtcactgtgaaa 91260 taagtcattc atttagccaa ggtgatcatt aaaaggtttt ttaaaagcaaaacctttatt 91320 atttgataga gaggagactc aattttctag tcaacagacc tgaaaaagacaatatgatac 91380 agaatctatc tctccttctt tcctctctct ttttttttgt gcagtttactcaaaaggtga 91440 acaaaaatat tttgctgtta ctgaagcttt ttatttgcct tttatagaaaatcttttaaa 91500 agagggaata aaaatattga aatcttatta gaagcttctg cacattaataggcatccgca 91560 tccttggatg aaactaagtt gggggccttt tttttttttt ttttttttgacatagggcct 91620 cactcttttg cccaggctgg agtgcagtgg cgtgaccatg gctcactgcgcctcagcctc 91680 tcaggcttaa gtgatcctcc tatgtcagcc tcccaagtgg ctgggaccacagggacttgc 91740 cactatgctc tgctaacttt ttttcttttt ttgtagagac aaaatctcactatgttgccc 91800 aggctagttt caaactcctg gactcaagtg atcctcctgc ttcggccacccaaagtacta 91860 ggattatagg catgaaccta gagccctcat ttgtaaatag acttcttaaagtgcagtatt 91920 attcattttg aatgttctac tataatttta aattacataa agtgagatttcaccatttca 91980 gtaagtgttt gctgctttag ggtcctaaca tttatgagtg tatagctaggcatagctgta 92040 aggtagaata ctcagttctt cagaaattaa ggatcccatt ttcccttgaatcttggcttt 92100 ggctgtcaga tcccattgat catccaatga tttttccatg cctaaacacacaagaaaaag 92160 aaacaaaggg cataggctgg gcgcagtggc tcacgactgt aatcccagcactttgggagg 92220 ccgaggcggg tggattacct gaggtcagga gttcgagacc agcctggccaacatggtgaa 92280 accccatctc tactaaaaat acaaaaatta gccgggcctg gtggtggacacctgtaatct 92340 cagctactcg ggaggcagag tcaggagaat tgctggaacc tgggaagcagaggtttgatc 92400 gctccattgc acttcagccc aggcaacaac agtgagactc cgtctcaaaaaaaaaaacaa 92460 aaaaaaaaac aaaaaaaaaa gagaaacaaa gggcatagac atagagcacaaaaatctctg 92520 tgaatttcca aaagccaaag ttcacacctt cttatttgcc attaactgccagtttcttcc 92580 tgactcagtt aaacatccaa ggcctctaac tgaatccaag tcagttaattatcagatcca 92640 gtctgattct ggacctggtc cagtttctgt catgacttct gaacccatttcagattttaa 92700 aatttgctca aacaaattca gataactcaa aacacaaatc catggagcttcagaatctga 92760 gagcttaccc acaatcccca gttgctgcaa gagaggaatg gacacagagagtctgactgg 92820 taccatgctt cgtcactcag tgcttctggg gattgctaga ggttctacttcggatcccac 92880 ttctgacacc atctgttaaa agaaaaacta gacaaattaa atttaacagagtttaattga 92940 ggaaacagtg attcacaaat caggcagccc ttagaaccag aataggttcaaagagactct 93000 ggcactacca catggttgaa gacttatgta cagaaaaagg aaagtgatgtacagaaaatg 93060 aaactgaagt acagaaacag ctggattgct tacagcttga tacttgccttatttgaacat 93120 ggtttcaaca gttggctgca ttttattggc tgaaactcag caattggtacaagaataggt 93180 tacagcttgt ttacacgtcc ggagattacc attcactatg tacagagaaacctttaggcc 93240 aaactttaaa aatgtaacga gacagcttta ggtgaaactt aatgtaacactacctaccag 93300 gtgaggatta actaagatcc ttatttttct gttgaggatg ttgaaggagtggttaaactc 93360 cttgtttcag tttccttact ttccaaaatt tgcctttcat ctccaccactttttctctct 93420 tcttcagatt ctgtccttca acttctaagt gtgcaaaaat cttcagttcagtttcttttt 93480 aatcagcaat tttcacatta cctaacaatc tcttaggctt ccagacctccattctctccc 93540 ctcccttgaa atcatagtgc tctgctaaaa ctggttcctg gaagatttcctgtaccttta 93600 tgcttagcaa gttcaatggt ctcttcttag ccattatttt tctcaaaatttgactgactc 93660 cttttttttt tttttttttt tgagatggga tctcactctg ttgcccaggctggagtgcag 93720 tgatacaatc acaactcacc gcagccttaa cttcccaggc tcaaacgatcctcccacctg 93780 agcttcctaa gcagctggga ctacaggcat atgccaccat gcccagctaattttgttttt 93840 ttgtatagat aggatctcac tatgttgcct agactggtct cgaactcctggacttaagca 93900 gtcctcctgc cttggcctcc caaagtgctg ggatcatagg tgtgagccaccgcactctgc 93960 cgaccttctt gacatcctcc tctcttgcct tctggttctt tagtttacgttttctggctt 94020 cttttccttt tcattatcta tctgctctcc tttgggattc tgacacagtctcaggggttt 94080 ctgctgtcac gcttgtttga gaaattcggc tgtcatgaaa gaacaccacctctatttgtg 94140 acgaaagcta ctctgaaatg tttagtcttc tctttgacta agagtgacattcaaaattag 94200 tatgacattt atttctttat tttattgaga cagagtctca ctctgtcatccaggctggag 94260 tgcaatggcg tgatctcggc tcactgcaac ctctgcctcc caggttcaagcgtaaaatca 94320 gtatgacatt tcatatcttt caccagtctc ttaagtcctt tatcattactccattttcat 94380 atattctggc caaagaaact gaagcatcgt attccttcta tctccctggcctttttaacc 94440 tgtttccact catgctgccc tttactgaga atgcctcctt cctatctctacccatcagta 94500 tcctctctat ttgtgcttgt cagtttttgt gcaaaataca attgtattataatgattttg 94560 taaatgtatc atccctctgg aatctaattt tcttgaagaa atgatccttatctaatttaa 94620 atctctactt atataaagta tgtcaacaat gaaacattct tgagtgatacagagaccagt 94680 ttacctcagg ccatttcaga atttgccttg ccttcttcat ggcaagaagaaattagaata 94740 tgagaaataa aatttttttc tatttaattc tgttcatccc tttttattaatcccaaatct 94800 ctaaatggat gctttaatga tcacttaatg tatttttttc ctccagagtttttatccccc 94860 tgctttcaag tgaaatgtca caaggatggc ttattacaat gctatgattatcttatttgg 94920 ccttgaaagg ccaaaaaaaa aaaaaaacaa aaccatttcc aatgtttttcaaacttgagc 94980 ttttttattt ttcattttat atttgttatt cgcagcttga atagatgtctcagaatcatt 95040 gggcttgtgg ctcttgttag gctaagtgaa aatactcaat ttcatacttctttaattaag 95100 caccgataag gaataggaga aacttcatct ctatcctgaa ggaatcagagtgttttgggc 95160 aaagatcact cagatccttg acttaggacc tcaattgttt taaaaaaccagtgaagcgat 95220 tgttgctcat tattgcttat ttatactgaa ttcaaaattt tctataaatatgaaattggt 95280 ggccgggagc agtggctcat gcctgtaatc ccagcacttt gggaggccgaggtgggtgga 95340 tggcctgagg tcaggagttc aagaccagcc tgaccaacat ggtgaaaccccatctctact 95400 aaaaatacaa aaattagcca ggcgcggtgg cacatgccta taatcccagctacttgggaa 95460 gctcaggtgg gaagatcatt tgagcctggg aagttgaggc tgcaggagtgagttgtgatt 95520 gtatcactgc actccagcct gggcaacaga gtgagactcc acctcaaaaaaaaaaaagaa 95580 attggttttg ggggtttttc agtgtaaatc aaagtacatt aaaatgctgcctgcctcatc 95640 tgtttatttg gagacaagag tctcactctg tcacctaggc tggagtgcagtggcataatc 95700 ttagctcact gcaacctccg cttcccagat tcaagtgatt ctcctgcctcagcctcccaa 95760 ggagcaggga ctacaggtgc ctgccaccac accagactag tttcgtatttttagtagata 95820 tgggttttcg ccatgttggc caagctggtc tcgaactcct gacctcaagtgatcctcctg 95880 cctcagcctc ccaaattgct gggattatag gtgtgaggca ctgcacccagcctcatctgt 95940 ttttaaattt tgtttttata ttttaaaaaa atcctctgag ggcataatctttcctgctat 96000 cctagtgtga gataggtaat actatagaaa ttctggctct accatttgtctaatcatttg 96060 aactttggcc aaatatgtaa tgtctatgaa actattttct gatctgtaaaacaggaataa 96120 tacctgccgt gcctctttct tatgaatctt gtgagatcaa attagaaaataaacaatagc 96180 taaaatgtat caagttttta ccatggtcct ggatggtgtg ctaagtgctttacatatatg 96240 atctcattta atcttcacac caaccctata gttgaagcaa tccttttacagatgaggaaa 96300 actgaagtaa gtgcctaaaa tttcatagta gtatgtagca gaactgggaatggaactctc 96360 catactaact ctagagctga gctcttaacc agcatatact attagtaatgctccttgtaa 96420 actacagaat gccgtataac tgtatagaat gttcttcatt tgcaaaatggattactgaca 96480 gaccattacg cttaacatca gtagtctggt gacctctcat atatgaagcacacaaatctt 96540 tgcttcatcc ttccattccc ttcccaaact tcccattact actttgtaggaattcatgac 96600 tagacaaagg ttttatattt agtggtttct ccttccaggg gtttcacagaccagctgcgt 96660 aagatttcta aggatgcagg gatgcccatc cagggccagc catgcttctgcaaatatgca 96720 cagggggcag acagcgtaga gcccatgttc cggcatctca agaacacatattctggccta 96780 cagcttatta tcgtcatcct gccggggaag acaccagtgt atggtaaggatatcttaaga 96840 ctgcattttt cctcaagtac ttgatgtcct tttaggatta tactgaaacatatcctaaaa 96900 ctttcaaata ttaaaatata ttttatgata cagtatttaa aaccatgtattattacttga 96960 agacaaatta atatagcaaa ctaaatagtc caagatgaga cattgtaaaaagagtttccg 97020 ggctgggcgt ggtggctcac gcctgtaatc ccagcacttt gggaggccgaggcgggtgga 97080 tcacctgagg tcaggagttc gagaccagcc tggccaatgt ggtgaaaccccatctctact 97140 aaaaatacaa aaaattagct gggcgtggtg gtgggcgcct gtggtcccagccactcagga 97200 ggctgaggca ggagaatggc gtgaacccag gaggtggagc ttgcagtgagccgagatcgc 97260 accggtgcac tccagcctgg gcgacagagc gaaagtccgt ctcaaaaaaaaaaaaaaagg 97320 gtttcagtag tgaaaagagg cattacataa caatggctaa agaaagaattattgaataaa 97380 aatggcattg agataatttg agtatcagta taatgtagtg gttaaaagcacaggctatca 97440 aattaaactg tgttgctcaa tattcaggta ctgccggttc tactacctgtgtgatttggc 97500 tgaattactt aatcacacta gcccttagtt tcctcatctc tggaattgggacaatattta 97560 tgtatgtatg tggtgtgtat gtatgggata ggatctctct cactctttcaggctggagtg 97620 cagtggtgca atcatggctt actgcagcct tgacctcttg ggctcaagcaatcctccttt 97680 ctcggcctcc caagtagctg gaactacagg catgtgccac cacactggactaatttttta 97740 tttttttatt ttttttgttt gtatttattt atttatttat ttttattatactttaagttt 97800 tagggtacat gtgcacaatg tgcagtttag ttacatgtgt atacatgtgccatgctggtg 97860 cgctgcacac actaactcgt tatctagcat tagatgtatc tcccaatgctatccctcccc 97920 cccccccacc ccacaacagt ccccagagtg tgatgttccc cttcctgtgtccatgtgttc 97980 tcattgttca attcccacct ataagtgaga atatgcggtg tttggttttttgttcttgcg 98040 atagtttact gagaatgatg atttccaatt tcatccatgt ccctacagaggacatgaact 98100 catcattttt tatggctgcg tagtattcca tggtgtatat gtgccacattttcttaatcc 98160 agtctatcat tgttggacat ttgggttggt tccaagtctt tgctattgtgaataatgccg 98220 caataaacat acgtgtgcat gtgtctttat agcagcatga tttatagtcctttgggtata 98280 tacccagtaa tgggatggct gggtcaaatg gtatttctag ttctagatccctgaggagtc 98340 gccacactga cttccacaat ggttgaacta gtctatttat ttttttgtagagacaggatc 98400 tcactatgtt tctcgggttg gtctcaaact cctgggctca agcaatccttaaaccttggg 98460 ctcccaaagt gcagggatta caggtgtgag ccactgcacc tagcctctttctggttttaa 98520 ttgagcattt tatatgattc tattttcttt cctcttttag tgtatcagttatacttcctt 98580 gatatatagc acacataccc tgggatacta ataataccta atccatatatagggttgttg 98640 taaggattaa ctgagtctaa tatgtaaagg gcctagaata gcacctgtcatatagtaaac 98700 agtcaatgtt aactgttatt attataaaca aaattttgtg agattaataagctaaatata 98760 aatcattgaa tcgtagaaaa atacaaagaa aatataattt tcaaaccactgaacggggta 98820 aaatcatgta agtttagaaa taatagaaga aatcacaaaa gaaaattgtcagatttgact 98880 gagtactgag taaacattaa attttctctt tctttctttt tttttttttttttttttgag 98940 agagagtctc gctctgtcgc ccaggttgga gtgcagtggt gcgatctcggctcactgcaa 99000 gctctgcctc ctgggttcac gccattctcc tgcctcagcc tcctgagtagctgggactac 99060 aagcacctgc caccatgtcc agctaatttt tttgtatttt ttagtagagacagggtttca 99120 ccgtgttagc caggatggtc tcaatctcat gaccttgtga tcctcccgcctcagcctccc 99180 aaagtgctgg gattacaggc gtgagccacc atgcccggcc cttttttttttttttgagac 99240 agagtctccc tctgttgccc aggctggagt gcagtggcat gatctcagctcactgcaacc 99300 tccacctccc aggttcaagc tgttcttctg cctcagcctc cctagtagctgggtctatag 99360 gcgcgtgcca ccatgcctgg ctaatttttg tatttttagt agagacagggtttcaccatg 99420 ttggccaggg tggtctcaaa ctcctgacct caggtgatct gcccaccccagcctcccaaa 99480 gtgctgggat tacaggcatg agccactgcg cctggcccct aaatttcctacatgtcacgg 99540 atgtactaaa atgaaaagaa aaccaataga gtgtggaaaa tatttgtagcaaatagaaac 99600 aataaacaaa tagtttatta ttagttaaat aaaaaccttc tttttttagattaatgagaa 99660 aagattataa actgaaattt agtaaaaaca aagcaagtaa atatatgataagtttgtttt 99720 tattcatagt taaagaaaaa tacaggccag atgcagtggc ttatgcctgtaatcccagca 99780 ttttgagaag ccaaggtggg cagattgctt gagttcagga attcaagactagtctgagca 99840 acatggcgaa acctcatatc tgcaaaaaaa tagaaaaatt agccaggcatggtagtacac 99900 atctgtggtc ctagctactt gggaggctga gataggaaga tcacttgagccagtaggtgg 99960 aggttgcagt gagccaagat catgccactg cactccagca gcctgggcaacagagcgaga 100020 ccctttctca aaaaaaaaaa aaaaagagag aagaaaagta caaatcaataaagggaaaaa 100080 ttttgtgctg ctaaattagc aaacatattt aaatattata atacctagtgcttgtgagta 100140 tgcagtgtat taatacgttg ctgatggtag tataaattga tattacctttttcatcaaaa 100200 ctgtaaacat gcctgggctg gtctcgaact cctggcctca agtcatcctcccgcctcagc 100260 ctcccaatgc tgggattaca ggcatgtgcc actgctcctg gcccctcctggttttaattg 100320 agcattttgt ataattctat ttttcattct cttttaacat attagttagacttcctttaa 100380 aaatttttgt agtgttttcc ctagactttt taatatatat ttacaactaatctgagtcta 100440 cttttaggta acattatacc acttcttaga tagtatagag ataccttgtaacagaatact 100500 cccaattttt cccttctgta ccttatattt ctgtcattta tttcacttatccataagcta 100560 taactaccca atacactgtt gctattacta ttttgaacaa ataatcatctattagctcaa 100620 ttaagaatat gaaaatacaa gattttattt tacctttatt tatttgctgaccttcaacct 100680 tcctccttac ttttgttttg ttttgttttt tagagacagg tcttgctatgttgcccatgc 100740 tggtcagtgg ctattgtcag gcatgaccat agtgcactgc agccccaaactcctgggctc 100800 aagtactcta cccacctcag cctccccagt agctggtact acaggcatgacccacactgt 100860 accctgcttt tccttcattt atgatttctg atctgtatca ttttccttctctctggaaaa 100920 ttcttttaat atttcttgcc aagcatttct accagcaaca aataccctgtttttatttgt 100980 ttgagaaact ctttatttct ccttcatttt tttttttgag ccacataatgatttaatgtt 101040 tacttgcaaa tcattcactc acataatttc aagtactaag tcattctggaattcttacat 101100 tctgacatta agaacattca catgttgtgc agccatcatc actatccatttccagaactt 101160 ttttccatca tcccaaactg aaactcttta tccattaacc aataactttaatatcctatt 101220 aaacctcacg taacccctgg aaagcactgt tctactttcc gcttccatgaatttgactat 101280 tctagtttcc ttatgtacat gagtcctaca atatttggct tttggcataatatcctcaag 101340 gttcatccat gttgtactat atgtcagaat tttcttcctt ttttgggccaaataatattc 101400 cattgtttgt atatgtgtgt gtgggggggc ggggggtgtg tgtgtgtgtgtatacaccac 101460 atcttgttta ttcttctgtt gatggacact tgggttgctt ctacactttagctatcatga 101520 gtaatgctgc tatgaacata ggtgtacaaa tatctcttca caaccctgttttcagctatt 101580 ctgggtatac atctaataag tgtcattgct gcatcatatg gtaattctatttttaatttt 101640 ctgaggaacc tccacagtgt tttccacagt ggctgcacca ttttacatgcccaccaacag 101700 cgcacaggag ttccagtttc tccacatcct tgccccaatg tttgttattctctggctttt 101760 tgatagtagc catcctaatg ggtgtgaagt ggtatctcat aatgtctttgattttgcatt 101820 ttcctaaatg attagagacg ttgggcatct tttcatgtgc ttatgggtcatttttctgtt 101880 tatcttcttt agagaaatgt gtattgtcat ttgtcctttt ttttaaggcaaggtctcact 101940 ctgtcaccca gactggagtg caatgacacg atcatagttc actgcagcctccatctccta 102000 ggcccatgca atcctcttgc ctcagcctcc tgagtagcta gaactaaaagcacataccat 102060 catgcctggc taattaaaaa aaaaatttgg cagggatggg gtcttgatttgttgcctaag 102120 ctggtctcta actcctgggc tcaagcaatc cgcctcagcc taccaaagtgctgggattac 102180 aggtgtgaga cattgcaccc atttttctat gttttttata tataagaagttatgctgggt 102240 gcagtggctc acacctgtaa tcccagcact ttgggaggcc aagacgggtggatcacttga 102300 ggtcaggcgt tcaaaaccag cgtggccaac atggcaagac cccatctctactaaaaatac 102360 agaaattggc tgggcgtagt ggctcatgcc tgtaatccca gcactttgggaggccaaggc 102420 gggtggatca cctgaggtca ggagttcaag accagcctgg ccaatgtggtgaaactccat 102480 ctctactaaa aatacaaaaa aaaaaaatta tcagggcatg gtggcaggtgcctgtaatcc 102540 cagctacttc aagaggctga ggcaggagaa tcacttgaac ccagaaggcagaggttgcag 102600 tgaaccgaga ttgcgccgtt gcactccagc cctgggcaac aagagcgaaactcttatctc 102660 aaaaataaaa aataaaaaca aaaaaaataa aaagtaaata aaaatacagaaattagctag 102720 gtgaggtggt gcacacctgt aatcccagct actcgggagg ctgaggtaggagaactgctt 102780 gaacccagga ggcagaggtt gcagtgagct gagatcgcgc cactgcactctaccctgggt 102840 gacagaatgt gactccatct caaaaaaaaa aaaagaagtt atggctgggtgcagtggctc 102900 ttgcctgtaa tcccaacact ttgggaggct ggagcaggag gatcacttgagcttaggagt 102960 ttgagaccag gctgggcacc atggtgagac ctccctcatc tctacttaaataaaaaaaaa 103020 aaggcttggt gtggtggtgc atgcctgtag ttccagccac ttgggaggctgaggcaagag 103080 gatcgcttta gctcaggagg tcgagactac agtgatgcat gatcatgccactgcactcca 103140 gcctgggtga cagagtgaga tcctctcaaa aaaaaaaaaa aaagttatatacaccgaaat 103200 atttatgatg gttataatgg cagaggagag gaaaggaaca ggggacttttgttctttgcg 103260 ctataatacc tctgaatttt taaattaaag ataagagaat aggattgaaagctgtaagtg 103320 tactgtgatt acaagtacat taaaataata taaatgagaa aattattggaagagagtact 103380 ataaaacgat aacaagggat tatgtgcatg tttcacttta tgcttttcagttattgcagt 103440 tatgctatgg caaaaatata cctttttata caaagaagaa aacaactagaaaaattgtca 103500 ctaaatactt aagctagtct tcaaagcagt atactttcaa ttttaacagcatcgccttag 103560 tactgtattg gcataatttt tggtatcctg taatacaatg atggattttaagggggaaac 103620 atagtagctt tattttcagt atgtaaaatt attttcaggt ctttttttctgactagaggt 103680 tttgcatcta ttccatagcg gaagtgaaac gtgtaggaga cacacttttgggtatggcta 103740 cacaatgtgt tcaagtcaag aatgtaataa aaacatctcc tcaaactctgtcaaacttgt 103800 gcctaaagat aaatgttaaa ctcggaggga tcaataatat tcttgtacctcatcaaaggt 103860 aagatatgct aatcgcttat gaaaatatta tttttatatc ttcatttgtctatatatgac 103920 catatctaac tactataagg gctgtgtaag agaccccctt aataattcctactatgggag 103980 attcgtagac attttggtaa aaaaataatt tggattggca aggttcagagattctcttgt 104040 gaaaatgccc cccacaattt tattttatct tattttttaa ggaaagaatgaagcaaagca 104100 acaaaagcag agatttactg aaaatgaaag tacactccag agggtgggagcaggctcaag 104160 ccccaaaaat tttcatttag tatgcaaagt acctcttacc tagccagaaagtatccagtg 104220 ctttatttct cactggagag cccagcttga agaattttca ctttatttggtaatcatctt 104280 caacctggtt tcatgagact actcacctac ctggaatgtg aaggagagaaggaactggga 104340 gcccagatta gtcaattaag taagccatat cagccttcct gctatagtcagtgagaaaac 104400 acattactgt tagatcaccc ttcttagagc tggagctctt aggaagtaagagacaacatg 104460 acctagaaac aggaggtagc ttattgtgca agtcactgag gaagaggtcatttttcagag 104520 aattgaacaa gttccctttt aatagttgaa gcaattaggc ctcaaaaaagttaactgtac 104580 accacaaata aacattagaa taagaattgg cccatgtttg tttgacacaaaagtcctttt 104640 tttcctgttt ttccatgcca tctctacttt ctacccctgt taaatatgagatgagatccg 104700 taaatgagat aaataaagaa ttttatcaac aagcccaaca tggtgaaactttatctctac 104760 aaaaaaatag aaagattagc catgtgtggt ggcatgtgcc tgtcgttccagctactccag 104820 aggctgaggt gggaggatta cctgagccca gggaagtcaa ggctgcagtgaactgtgatt 104880 acgccactgc actccagcct gggcaacaca gtgagaccct gtctcaaaaaaacaataata 104940 acataaagta aaataaaagg aagtcctagc ttcctttatg accattgaatatatgttact 105000 caaatttata cttttggcca gggagtggga gtagtggatt cccaaaatcccttccagtct 105060 gaagtgtaat gattcctttt tcccttgaga gattacatag gtttaaaatataagtctttt 105120 tttaaagagt gataaaaatt tgtggatcat aaggaagttt ataattttgtgcccatgttg 105180 gagaatacta gcatctgtta atattaatac agcccttttc aaccaccagcaccactattt 105240 tagataaaat tctgctgtga ataaaacttt ggattggccg ggtgcggtggctcacgcctg 105300 taatcccagc actttgggag gccaaggcag gtggatcaca gggtcaggagatcgagacca 105360 tcctggctaa catggtgaaa ccccatctct actaaaaaat acaaaaaattagccgggtgt 105420 ggtggcgggc gcctgtagtc ccagctactt gggaggctaa ggcaggagaatggcatgaac 105480 ttgggaggtg gagcttgcag tgagccgaga tcgtgccact gcactccagcctgggtgaca 105540 gagcgagact ccgtctcaaa aaaaaaaaaa aaaaaaaatt tggattacataggacttcct 105600 ctgtgctgtc aataacttga agttacgttg caattgtgtg aaacagataaatggatactg 105660 aaaaaatgaa atatctaacc ctttttcaaa atgtgtttaa gaccttctgtgttccagcaa 105720 ccagtgatct ttttgggagc cgatgtcact catccacctg ctggtgatggaaagaagcct 105780 tctattgctg ctgtgagtgt tagccaggtt tatcttacct aaggttgacagaccagtatc 105840 aattttgcag tttcaacttt ttgaatttta atattttctt tgtagccaactttcataaat 105900 gttctttggg tttttgaaaa gaatgattat tctctggttt ggagtttggaagttccatat 105960 gtatctatta aatcaagttt gctagttata ttattcaaat cttctgtattatactttaat 106020 ttgtctactt gacctctcca tgattatgca tttttagtta ctttttgttgttttgaatag 106080 tttgtttgta tatttcagta atgtctttgt catagaaaga tacactacatataagcttgg 106140 tggattacat tttttatata taaaatgtct cccaccttca tccaggtcaatactttgcac 106200 ctgaattctt ttattttatt ttattttatt tatttttttc agacaaagttatcttgttgc 106260 ccaggctaga gtgcaatgac acaatctcgg ctcactgcaa ccttcatctcccaggttcaa 106320 gcgattatcc tgcctcagcc tcccgagtag ctgggattac aggcacctgccaccatgccc 106380 ggctaatttt ttgtactttt agtagagacg gggcttcacc atgttggccaggctggtctt 106440 gaactcctga tctcggtcag tccacctgct tcggcctccc agagtgctgagattacaggt 106500 gtgagctacc gtgcccagcc aaattcttta ttttactact gccactcctgtttttaacat 106560 ctgcctgtta tttctttgct ctctgtgttt gtaacctttt tgtctcagtttaagtgtatt 106620 tcttataaac ttgagaaagc ttgataatat tttacctatt ttgaaaatcacttgatttta 106680 acaaatagcc aacatttcga aaatatgaac atcttttagg catatttaataaacttcaga 106740 aattaaactg taagatttaa ttggccaggc gcggtggctc acgcctagaatcccagcact 106800 ttgggaggct gaggcggggg tggatcgctt gacgtcagga gttcaagaccagcctgacca 106860 acatggtgaa accccatctg tataaaaaat acgaaattag cctgtcgtggtggtgggtgc 106920 ctttaattcc agctattcgg gaggctgagg caagagaatc acttgaactcgggaggcgga 106980 ggttgcagtg agctgaaatc atgccactgc actccagcct gggtgacagagggagacccc 107040 atctcaaaac aaaaaaaaag atttaatatt ttttctatga tacattataatcttcacata 107100 aatcttgttt tatgaatctg ataaatctaa ttcatccctg aaaatgatagtaatatttaa 107160 atttccactt catgatttca aaactatttt cattagtaaa ggaaattaatttttatctat 107220 taattctctt gcttttgtat ttttaatgtt atacatgaat tattttctttaaatgttcag 107280 tggttgtatt gagtcatctc taacattcat taaagtgttc ttatagatgataaaatgata 107340 aagaaaggaa ctcaaaagac ttccttggat ttgctacttt gggtcattttccacaaaggt 107400 ttaagcatat tttaattcct ttggacttct ggtagtcatt gtctttttctactaaatccg 107460 tttctaccaa atacccactt tcttttgtgg ttctcagaag tcttgtaaatagaatttatt 107520 ggttgattct ttttcactat atggcttcta tctccaagaa cttagagagtagtgtgagaa 107580 actaagtaag taatgagtgc cacatagtat aataggtgtt atcatgataacgaagtaaca 107640 acatttaagt tgcaagaacc tggtgtgttt tgggaatgtt gagaaatacaaggaattgga 107700 acatagtata ctgcaggcta tgaggctagg aagcctttgt gaggcattttgttccatccg 107760 agggtctgga tttattcttt tttttttttt tttttttgag acagagcctcgctctgtagc 107820 tcacgctgga atgcagtggg cgcgatcttg gctcactgca agctccgcctcccgggttca 107880 cgccatcctc ctgcctcagc ctcctgagta gctgggacta caggtgcacgctgacacacc 107940 cggctaattt tttgtatttt tagtagagac ggggtttcac tgtgttagccaggatggtct 108000 tgatctcctg accttgtgat tcgcccacct cggcctccca aagttctgggattacaggcg 108060 tgagctaccg tgcccggcct ggatttattc ttaagtagcg agatacctttcaagggaaag 108120 aaatggtcaa agttccattt ttgaagcatg actttggtat aggttaactggaagaaaggg 108180 tctggaggca gtgaccagct ctaggctctg atcagaataa tgttataagttcatgctttg 108240 aggtctcttc tccaaagaca taacaatatt agacacagaa agagaaattaatatagctag 108300 tttcaaaacc aagattaact tctctgagga ccagatacag aacacaaatgcagctcaata 108360 agactgtata gattcaggcc aagagggaaa attctgattt taaaatttatgaagcgtaac 108420 agttctaaga tgaagctaaa gtcttaagta tggcttctga gataggttgctaaagtggaa 108480 gagataggaa gaaaaatcag tggtttgaat tggtccttgt tttttcttatttcttttctc 108540 ttattctctt atacacatct ttttgtcttt ttctttcctt actaatctttaagtctacca 108600 acaacacaaa gaatctaatt gttaaatgta tagttcagcc tagttataggaaaatagttt 108660 tttaccctat actaggtgtt ttattctgtt cttcaacggg aagtactattctttttaatt 108720 ttttaaaaag atggggtagg ctgggtgcgg tggctcatgc ctgtaatcccagcactttgg 108780 gaggccaacg cagacggatc acgaggtcaa gatatcaaga ccatcctggccaacatggtg 108840 aaaccccatc tctactaaaa atacaaaaat tagctggggg tggtggtgcacacctgtagt 108900 cccagctact ctggaggctg aggcaggaga atcgcttgaa cccaggaggtgcaggttgca 108960 gtgagccgag attgtgccac tgcactccag gctgatgaca gagcaagactgcgtctcaaa 109020 aaaaaaaaaa aaaaaaaaaa agagagaaag agagagagat ggggtcttgctattttgccc 109080 aggctggcct taaattcctg ggcacaagtg atcctcctgc ctcagcttcctgagtagctg 109140 ggactatagg cacatgccgt catacccaac tcagaattac tatttttgattactttcaaa 109200 gtaaacagtg atgaacaggt tacagtaaaa taaaaatgta aatgaaattcatgtaatttt 109260 gattatcatt tatttttatt tatttattta tttattttac tttttctaacctaggttgta 109320 ggtagtatgg atgcacaccc aagcagatac tgtgccacag taagagttcagagaccccga 109380 caggagatca tccaggactt ggcctccatg gtccgggaac ttcttattcaattttataag 109440 tcaactcggt tcaagcctac tcgtatcatc ttttatcggg atggtgtttcagaggggcag 109500 tttaggcagg ttggttacct agaatctcat cagactatgg tgaaatcagatattgtgttt 109560 ataatatggt gtctagttct agagttaaaa accttgttag agttccccaagtcaaaactt 109620 ggtgttttgt tagattgtct ttttgaaaat gttcactacg aatgtgtatgccttgcttgc 109680 taagaccatg ttctaataat cgttaaaatg gaatctatta gctatagagtggtgaaatta 109740 aggaatcctg agattaaatc attatcctac tttgaagtat ataaaaacaaatagctgact 109800 atattgaatc tttccatttc atattctcta ggtattatat tatgaactactagcaattcg 109860 agaagcctgc atcagtttgg agaaagacta tcaacctgga ataacctacattgtagttca 109920 gaagagacat cacactcgat tattttgtgc tgataggaca gaaagggtaatctcacctct 109980 gttgtaatac tgttataaac caagcttatc caacatagtt catagagcattcaacaaggg 110040 ccctctgcca acagcaggat ttccaatata tagtagcaaa tttcaacaattacattatga 110100 gtatagaagc atcaaaatag attattttta actttctaga atttctagtcacactccaaa 110160 tattcaaaat ctcattctac ttttttgttg tgaagaaact catggttttaaaattattct 110220 tctattcata ttttcctaaa atcttcagaa aaaacagtac attctctttttaaaatatct 110280 catacaaatt agttttttaa aaaagtggaa attcaggcca ggcacagtggctcacacctg 110340 taattccagc accttgggag gccaaggcag gcagatcact tgaggtcaggagttcaagac 110400 cagcctggcc aacacggtga aaccctgtct ctactaaaaa tacaaaaaatagccaggtgt 110460 ggtggcgcac atctgtaatc ccagctactc gggagactga ggcaggagaatcacttgaac 110520 ccaggaggca gaggttgcag tgagccgaga tggtgccact gtactccagcctgggcaaga 110580 gagcaaaact ccatctcaaa aaaaaaaaaa aaaaaaaaag tggaaatttatacaaactaa 110640 cttttagcca tgtttccttt aaagttcaca gaattatttt atgcattttataatgtgaat 110700 gtatattttt tataattagg aaggaaagag aggtataaat acctaaggaaatatgttttc 110760 tttttttttt tttttttttt ttttgagacg gagtctcact ctgtcgcccaggttggagtg 110820 cggtggcgcg atctcagctc gctgcaagct ccgcctccca ggttcacgccattcttctgc 110880 ctcagcctcc tgagtagctg ggactacagg cgcctgccac catgcccggctaattttttg 110940 tatttttagt agagacaggg tttcaccgtg ttagccagga cggtctcgatctcctcactt 111000 tgtgatccgc ctgcctcggc ctgccaaagt gctgggatta caggtgtgagccactgcgcc 111060 cggccagaaa tatgttttct tatctgtgtg cagcaagaca gcagtcttactgtcattttc 111120 agtgctctga tcacatctgc ccccatcctg taaatcttgg agcagagaaatctaatgata 111180 ccaagtgttg tttccccttg agaaagggat ttgatagaaa ggactaagagaggggtgttt 111240 ttctttgcca tacatactca ctgtattcct ttgggcaagc aacttaacatctctgtgtct 111300 gtttatttct tagcaagatt ggataatatt aatatctacc ttatttgggttgtcgggaga 111360 attaaataaa atacgtaatg cacttagaac agggcttggc ccacactaagtactcagtaa 111420 agattggcta gctctcatta gtagagtgat gagaacaaaa aaaaattaataaataaagtt 111480 tggctagctg cagagtggtg gtgatagtgg tagaatagag aagaagaaataataatagat 111540 attgttctaa gtattgctta taaatttcat caatttaatc ctcaccaacaagattatgag 111600 atagttgtat ttttttgttt ttgttttttt tgacagtttt gctctgttgcccaggctgga 111660 gtgcagtggc atgactttgg ctcactgcag cccccacctc ctgggttcaagtgattgtca 111720 ggcctcagcc tcctgagtag ctgggattac aggcatgtgc caccacacccggctaatttt 111780 tgtagtctta ttagaggcag gatttcacca tgttggccag gctggtcacaaactcctggt 111840 ttcaaatgat ccgcccacct cggcctccca aagtgctagg attataggtgtgagccatca 111900 cacccagcct acaacttttt gtatatatta attagaagat ttacgtgttgctaatttatt 111960 agagttaagt atttttttag gaccaggcat aatttagtag attatgaactgttttgtttt 112020 gtgcttggta aagtgaagct ggttggtcca caccactata attcaaggtactaccttata 112080 acagtcactc acgtggcaaa aatattatct gagtcagtaa aatagtttctgatttgctag 112140 ccttgtaaac ctagacttac agttaatgat tttaaatgtg ttgggataaggcggtggagg 112200 tgggtataat aaacattttt aagtgtttta taaggagctt gtatcatcaagaattttggg 112260 tcatttcagt tatattatat ccactcagag aaaatcatgg tgtcctcttacaagatgttg 112320 aaactgatag tatgaaataa aaggaataag gcctttgcaa ttatttcattgtttgaatcc 112380 cagttctgcc ctttattgtg tgctattgag caaattattt tgtattcctgagcctttatt 112440 tccctaaaca aatggaaata atccctaact tgcaggattg ttaccaggttttagaaataa 112500 tgtgttcaaa gtgcctgatc cataaatggt taacaaatgg tgtgactgttgttgatcttg 112560 tcagcattct ctctctctaa acatctagtt ttctccaagg taaatgctgatcttcataaa 112620 tcacacgttc aagaatacct actatgcaca ggtaccataa attggagcactttaagttaa 112680 tttaaaaaaa atttttttta gtgtcttcca cccatagtgg ggtcaactatcagctaagca 112740 gtagtagaga ctgtattagg ttttgttgtg tatcagcgct cactttcagcaacatgtaag 112800 aataggaact aggacctgcg tagtctgctc tgaatgactg cttaagtcatatttcaaggt 112860 gaaagtagtg aaatgtagga tcagcctatg tattcataca ttttttaaagtgctttcaga 112920 tatattttta cctttttctt gtttgtttaa actttaacca aacaaatctaggttggaaga 112980 agtggcaata tcccagctgg aacaacagtt gatacagaca ttacacacccatatgagttc 113040 gatttttacc tctgtagcca tgctggaata caggtaagcc tacactttgggtaaaatatt 113100 ttaattcaag aactgtcatt cttacgtgta ttttttaaat ctcagaaaaaggataaagaa 113160 atactctttg cattccaaat tgtttccaca tgaaattaga tgaaactttcagtaaacaaa 113220 tgtcttccct ttccttacct tgagaggggc ttaggaactt atttttatgaaaataccaga 113280 atataagtag tttaatagaa tgaatctccc taaaacaggc ccttaatttcactaaaatgt 113340 taaaaaatgt gaaagtacct gattgttcct ttagcatatg catctttaaaaaaaaaagag 113400 agagagagaa agagaaagca ctattttgct caggctagac tagaactcttgggctcaagc 113460 agtcctccca cctcagcctc ccaagtagtt gggacaacag gcctccacactcagctaatg 113520 acgataacat tttatagaaa actttgacat gtactagtag tattaagcatgtgaaagagt 113580 ttaattgggc tgggcatggt ggctcacgcc tataattcca acactttgggaggctgagat 113640 gggcagattg cttgagttca ggagtttgag accagcctgg gcaacatagagaaaccccgt 113700 ctctacaaaa aatataaaaa ttagccaggt gtggtagcac gtgcctgtagtctcagctat 113760 tcaggaggct gtcgtgggag gatcacttga gcgcggaggc agagggaggttgccatgagc 113820 caaaatcatg ccactgcact ccaggctagg tgacggagcc agaccttgtctcaaaaaata 113880 aaaaaaaatt taattgtaga attacagctt tagttttttt ttgttgttgttgttgttttg 113940 ttttgtttgt ttgtttgttt gtttttgaga cagagtctca ctctgttgcccaggctggag 114000 tacagtggca tgatctcggc tcactgcaag ctccgcctcc caggttcaagtgactctcat 114060 gcctcagcct ccctagtagc tgggattaca gactgagatt actgggtgccaccacaccca 114120 gtaatttttt tatttttatt ttttctagta gagacagggt ttcaccatgttggccaggct 114180 ggtctcaaac tcctgacctc aagtaatccg cccaccttgg cctcccaaagtgcggggatt 114240 acaggcttga gccactgcac ctggccttaa gtttcagtta aataatttaaaaagtttttc 114300 ttattctttc cagaaaacct tttcattaat gtgttagctt tacttggtggaacataagct 114360 ttaaatcagt gcatctcaaa ctttccaaca acaaaaagta aactgacattcccaggatgt 114420 ctgagagtct ataacccaaa actattttag gttacttgcc attattgattaagatagaaa 114480 tttccagatg taccttacca tacagctctg tatcttatag ctttatatgtcccacatata 114540 cgaacctgtt tcagattcat ggctttaaat tattgtctga ctcatttatccatgttctta 114600 tatcagctcc tctttacaat taatgttgta cattttcagg gttggtggttaatgggaggt 114660 ccagaggaag tgatgtcagc aagatggctg actagaagcc cctagtgcttgctcccctca 114720 caaagaaagc cagaaaaaca gataaacaac tacatttttt ttttttaacggagtctcact 114780 ctgtcaccca ggctggagta cagtagtgtg atcttggctc actgcaacctctgtctccca 114840 ggttcaagca attctcctgc ctcagcctcc caagtagctg ggactacaggcacccaccac 114900 catgccgagc taatttttgc atttttagta gagatgggat ttcaccatgatggccaggct 114960 ggtgtcgaac tcctgacctc aggcaatccg cccacctcag cctaccaaagtgctgggatt 115020 acagacatga gccaccatgt ccgacctttt ttttttttga gatggagttttgcttttgtc 115080 gcccacgcta gagtgtaatg gcgtgatccc aggctagagt gtaatggcgtgatctcagct 115140 cactgcaatc tccacctccc aggttcaagc gattcttctg cctcagcctccggagtagct 115200 ggaattatag gcttctgcca ccacacgcgg ctaattttta tatttttagtagagatgagg 115260 tttcaccatg ttggccagga tggtcttgaa ctcctgacct ccggcgatccacttgcctca 115320 gcctctcaaa gtgctgggat tgtaggcatg aatcactgca ctgggccaaacaactatatt 115380 ttaatgaaaa taactgaggg agagccctgg agtgcatcag aggagtaacagaaaccctgg 115440 tgagcacaga aactcgggat gaccacacag agaacagaaa gaaacactgagcctccacta 115500 ctccatctcc aaatcaggat cagctgggaa ccaggaggaa cttctccctgcagtgaacag 115560 ataagcaaga ggatcccagc aatccccatc aacaccttgg acacctacactggggtcccc 115620 agcactgttc ttaggcacta atcccatctt ggggagttgc ctggagtccacataagtgta 115680 ccctcccaac ccgagaaaag gagctgatac tgtgctccac ccactgtggtccaagcagct 115740 actgcactac tccatcttgg aagtggaact atagctggga tatgtcttgctccaggggca 115800 agtagccatg actcctcttt attgttaagg ctatgctatc accaaattactccagcccag 115860 tggcctgaca gccctgtcga gctgcaagca cctgttacac cttgtgccttggccatttaa 115920 agagatcata cctcggccag gtgcggtggc tcacacctgt aatcccagcacttcgggagg 115980 ccgaggcggg catatcacct gaggtcagga gtttgagaac agcctggccaacatggtgga 116040 accctgtctc tactaaaact acaaaacaaa ttagttggac gtcatggcgcgcgcctgtag 116100 tcccagctac tccagcctgg gcaacagagt gagactcagt ctctatttaaaaaaaaaaaa 116160 aaaaaaaaaa aaaaaaaaaa gaggtcatac ctctccagtg cctaaattgaaggagtacat 116220 tgcatctcaa tgtactaagc agtgccttag tcatccagag cagtcacacactccagtacc 116280 taagctgaag cagtgccctg catatcaggg aaaccatgtc tgggccacccagaacaggca 116340 tagccccatg cctgagctga actggcactg gggaattggt gccctggaagatctgagcag 116400 ctctgtatcc catagatatt gggatagccc aaatatattg agatacaagaaaataccaat 116460 agacaactca acaaaatcgg gaaagcaggc cggccgcagt ggctcacgcctgtaatctca 116520 ttactttggg aggccgaggc aggcagaaca cctgaggtcg ggagttcaagaccagcctga 116580 ccaacatgga gaaaacttgt ctgtattaaa aatacaaaat tagctggggtggtggcacat 116640 gcctataatc ccagctactc gagaggctga ggcaggagaa ttgcttgaacctgggaggtg 116700 gaggttgcag tgagccgaga tcacgccact gcactccggc ctgggcaacaagagcgaaac 116760 tctgcctaaa aaaaagatcg ggaaagcaat tcacaatatg aacaagaaattcaacaaaaa 116820 gatagaaatt taaaaagaac caaacagaaa tcttcagctg aagaattcaatggaaaatac 116880 aaaatataat agagagcttc aacagcagat ttgatcaagc agaagaatctctgaacttga 116940 agacaggtca tttgaaaaaa aaatcggagt cagaaaaaaa aaagaagtaagaatgaaaaa 117000 gagtgaagag cgcctctgag acttatgaaa caccagtaag caaataaatatttgtattat 117060 gggagttcca gaaggagaag agaaagggaa aggtgtagaa aaatctgcttaatgaaatat 117120 taggttgggc atggtggctt acacctgtaa tcccaacact ttgggaggccaaggtgggca 117180 gaccgctttt gctcatgagt tttgagtcca gcctgggcaa catggtgataccccatccct 117240 acaaaaaata caaaaattag ccaggcatgg tggcatgcac ctgtagtcccagctactcgg 117300 gactgaggca ggaggatcac ttgagccggg gaggttgagg ctgcaacgagctgagatcac 117360 accactacac tctagcctgg gatgacagag tgaggccctg tctcaaaaaaaaaagaaata 117420 atagctgaaa acttcccaaa tctggggaga aatatagaca tctagatctaggaggctcaa 117480 aagtctccaa acagatcgaa ccctaaaagg ttcttcccaa ggaacattgtagtcaaattg 117540 tcaagtcaag gacagacaga attctaaaaa caacagaaaa gcatcaagtcacttagaatc 117600 ttcattaaac taacagcaga tttctccaca gaaaccttat aggcctggagagattgagat 117660 gatatattca aagggctgaa aaaaaaaatt gtcagccaag aatgctgtacccagtagagg 117720 atcatgtgag ctcaggaggc agaggttgca gtgagctatg attgtgccactgtactccag 117780 cctgggcaac ggagtgagac cctgtctcaa tcattcaatc aatcaatcaatcaatcaata 117840 agaatcctat ttccagcaaa gctaactttt agaaatgagg gagaaatagtatttccgaga 117900 catgcataaa ctgaggacat ttatcaccac tagattcgac ctataggaaatgttcgaggg 117960 aggcaaaaag tcaataatca ttattatgga aacaacagta taaaactcactggtagaaca 118020 gatacacaag aaagaaacag aaaagattca aaccttgttg ctctacagaaaaccaccaac 118080 ccaccatgat aataagaaga aaggaagaaa ggatatagaa aacaagcagaaaacaattaa 118140 caaaatgaca ggaataggcc ttcacctatc agtagtaact ctgaaggtaaacaaattaaa 118200 ttgcccactg aaaagatgta gactggctaa atgaattttt ttctcactctggtttccagg 118260 ctggagtaca gtggtgcaat ctcaactcac tgcaacctct gcctcctgggctcaggcgat 118320 cctcttgccc cagcctcctg catggctggg actacaggca tgcatcaccacacaaggcat 118380 gcatcaccac acctggctta tttttgtatt tttgtagaga cgggatatcactatgttgcc 118440 cagactggtc ttgaatccag agctcaagca atccacctgc ctcatccttccaaagtgctg 118500 ggattactgg cgtgagccac caagcccagc cactgaatga attttttaaaagaatgaaac 118560 catatccttt acagcaacat agatggagct gaaggtcata atcctaagcaaactaacaca 118620 ggaacagaaa accaaatact gcatgctctc acttaaaagt gggagctaaacattgagcac 118680 acatggacat aacatgggaa taataaacac tgtggactac tagagaggagggcagggggg 118740 atgggttgaa aaattctatt ggggctgggt gcagtggctc acgcctgtaatcccagcact 118800 ttgggaggcc aaggccggtg aatcacaagg tcaagagatc aagactgtcttggccaacat 118860 ggtgaaaccc catctctact aaaaatacaa aaattagcca ggcatggtggcacgcacctg 118920 tagtaccagc tacttgggag gctgaggcag gaggatcact tgaactcaggaggcagaggt 118980 tgtagtgagc cgagattgcg ccactgcact ccaacctggc gacagagcaaggctctatct 119040 caaaaaaaaa aaaaagaaaa gaaaaactac tgggtgctaa gctcaccatctgggtgtaat 119100 atacccatgt aacaaatctc acatgtacct cctatattta aaatactgaattttttaaca 119160 atccaaatat atgctgccta tgagaaattc acttcacctg taaagacacatataaactga 119220 aagtgaagag atggagaaag tcattcattc catgcaagcg gaaaagaaatgcaagcagga 119280 gtagctgtat ttaagttaga caaaacagac tttaagtcaa aaactataaaatgagacaaa 119340 gaaggtcatt atataatgct aaagggatca atttaccaag aggatataacagttgtaaat 119400 atatatgcag ccaacactgg agcatccaga tatataatat aaagcaagtattattagctc 119460 taaagaaaga aggagactcc aataatagta gttgagaact tcaacaccccactgtcagcg 119520 cttgacagat catctagaca gaaaatcaac aaagaaacat tggatttaaaatacacttta 119580 gaccaaatgg acctaacaaa tatatacaaa gcatttcatc cagcagctgcagaatataca 119640 ttattttctc agcacatgga acattcacca ggatagacca catgttaggccacaaaacaa 119700 atttcaacaa atttaaaata atggaggcgg tcaggtgtgg tggctcatgcctataatccc 119760 agtactttgg gaggccaagg tgagtggatc acttgaggtc aggagttcaagaccaccctg 119820 gccaacatga tgaaacccca cctctactaa aaatacaaaa attagccaggagtggtggca 119880 catgcctgta atcccagcta ctcaggaggc tgaggcaaga gaatcgcttgaacctagaag 119940 gtggaggttg cagtaaacca agatcacgcc actgcactcc agcctgggcgacagagtgag 120000 actgtctcaa aataaataaa ttaaattaaa taattgccgg gtgtggtagctcacgcctgt 120060 aatcccagca ctttgggagg ctgaggcagg tggatcactt gaggtcaggagttcgtgacc 120120 agcctggcca acatggcaaa accccgtctc tactaaaaat acaaaaattagccgggtgtg 120180 gtggcgggcg cctataatcc cagctactca agaggctgag gcaggagaatcgcttgaacc 120240 caggaggcag agattgcaga gccgagatca caccattgca cactccagcctgggtgactg 120300 agtgggactc tatctcaaaa aaaaaaaaaa aaaaagatac ttgatttgatttctttcgtt 120360 ttgttttgtt ttgttttgtt ttgttttgtt tgagacaggg tctcactctggtgagatcat 120420 ggctcacagg aaccttcgcc tcctgggctc aagtgatcct cccacatcagcctcccgagt 120480 agctagggcc acagacacat gccaccatgc ctggctaatt ttgacattttttgtagagac 120540 aaggtttttc actatgttac ccaggctgag tttaagcagt ccacctaccttggcctccca 120600 aagtgctggg attacagatg ttagccactg cacccagcct caattatttttttgatttaa 120660 tgttggccag gctggtctcg aactcctgac ctcaaatgat ccgcctgcctcggcctccca 120720 aagtactagg attacagtca tgagttacca tgcccggcct caaatatcttttacatggaa 120780 attaaacagt atgctcctga acaaccagtg actggtcaat gaagaaattaagaagaaatt 120840 tttaaaattt cttggaacaa atgaaaatag aaatacaaca ttccaaaccctgtgggatag 120900 agcaaaaaca gaattcagag ggaagtttat agcaataaac atttacatcaaaaacataga 120960 aacggctggg cacatggtac atgcctgtaa tcccagcttc tcaggaggctgaggcaggag 121020 gactgcttga gcccacaagt tcaagacaag cctgggcaac atagtgagacctcatctcta 121080 caaaaaataa aaaaattagc tggacgtgct gacatgtacc tgtggtcccagctactcagg 121140 aggcagaggt gggaggatca ctggagccca gaagggcaag gctgcaatgagcctgggcaa 121200 cagaacaaga ctctgtctca aaaaaacaag gtattaatat ccaggatatccaaagaactc 121260 aaacaactca acagcaaaaa taataataat aatttgattt taaaataggcaaatgtctca 121320 agagagtaat tgagacgatc aagaggtata tgaaaaaatg ctcaacatcaataatcacca 121380 ggggaatgcc aattaaaatc acaatgagat atcatctcac tcaaattaatatagtcatta 121440 tcaaaaagac aaagaatgca gagaaagggg aactcatatg cactattggtggcaatgtac 121500 attagtacag ccattaggga aatcagtatg gaagttcctc ataaaactgaaattaaaact 121560 accatatgat ccagccatcc cactactgag tatatatgca aaggaaaggaaacagtgtgt 121620 caagaagata tctgcatccc catgtttatt gcagtactat tacaataggcaagatatgga 121680 atcaacctaa gtgtccctca acagatgaat gaagaaaata tggtatttatacacaatgaa 121740 atactattta gccacaaaaa aagaatgaaa ttctgtcatt tgtggcaacatggatgagtc 121800 tggaggacat tatgttacat gaaataaacc aaccacagag agatatatactgcatgatct 121860 cactggtgga agctataaaa gttgatgtca tagaagtaga gagtaaaatagtagttacta 121920 gaggcaggaa agggaggggg attaccaaag actggttaac agatacaaaattacaactag 121980 ataggaggaa taagttctaa tattctatag cactatagga tggctgtaattaccaactta 122040 ttgtatattt tcaaataacc aggagagtgg gtttggaatt ttcctggcacaatgtttgag 122100 gcatagatat cctaattacc ctgatttgat cattatacat tatatatgtgtatcaaaata 122160 ttacactgta cctcataaat atgtgtaatt attatgtgtc catgaaaaataacaaaagcc 122220 aaaaaaagtt aatagtatct tctagggatt aaaacctata tatatcaggctcaggcctgt 122280 aatctcagca ctttgggagg ccgaggcgag tggattgctt cagcccaagagttggagacc 122340 agtctgggca acatggcaaa acctcatctc tataaaaaat aaaaaaattagctgggtgtg 122400 gtggtaggtg cctgtagtcc cagctactcc ggaggctgag gggaggatcacctgagcctg 122460 ggaggtcgag actgcagacc ctgtctctaa ataaataaat aaataaatcctatatgactg 122520 gtttatatat tttaacctag tgtttcctcc agtatttaat aaataccacattgcatccag 122580 atgactgtat tttaaaaatc acagaggaga aagattctct cttcaagttaagttttagaa 122640 atgctgttaa acagattttt tttattacgg aacttttgaa gaccattgatatgcaaatga 122700 ccattatgaa tcttcaagat agaaatattt ctccgatata tttaatcagaaaccttaggg 122760 aacagttttc cttttttttt ttaactttta aaatttttat ttttatttatttatttactt 122820 tttgagatgg agtctcgctc tgtcacccag gctggagtgc actggtgcagtcacggctta 122880 ctgcaacctc cacctcccag gttaaagaga ttctcctgcc tcaccctcccaagtagctgg 122940 actacaggca catgccacta cacccagctc attttttgta tttgtagtagagagggggtt 123000 ttgccatgtt gggcaggctg gtctcgaact cttgttcgat ccacctaccttgtcctccca 123060 aagtgctggg attacaggtg tgagccacca cacccagcct atttttgttttattatttat 123120 ttgagacagg gtcttgctgt gttgcccagg ctggagtgca gtggcacaatcaccactcac 123180 tgcagccttg acttcctgag ctcagcaatc ttcctgcctc agcctcccacctagctggga 123240 ctacaggtcc acaccaccac acctggctaa tttttttttt ttttttttttttttgagaca 123300 gtctcgctct gtcaccaggc tgaagtgcag tggtgcgatc tcggctcactgcaacctcca 123360 actccctggt tcaagcgatt ctcctgcctc agcctcctga gtagctgggcttacaggcac 123420 gcatcaccac gcctagctaa tttttgtatt tttagtagag atggggtttcaccatattag 123480 ccaggatggt cttgatctcc accacatctg gcccacacct ggctaattttttaaatcttt 123540 ttgtagagat gaggtcttcc tatgttgcct aggctagtca caaactcctgggcttgagct 123600 gtcctcccac ctcagcctcc ccaaagtgct gagactataa gcatgagccaccatgcccag 123660 cctgaacaga tttcctttag gatatcagtt tgaaacttct gctgtagtcagcaaataacc 123720 aataatattt cacagagact aaaaactgtt ctttatacat cttcccagctttacttttct 123780 attttatttt atttcatttc atttatttat ttattgagac aaagtttcactatgtcaccc 123840 aggctggagt gcaatgatgg gatcatagtt cactgcagcc ttgacctcctgggctcaagt 123900 gatctgcctg cctcagcctc ccaagtagct gggactacag gtgcatgccaccacatctgg 123960 ctaatttttt ttttaccttt tgtggagatg gagcctcact gtgttgtgcaggctggtctt 124020 gaactcctgg cctcaagcta ccctcccact ttaacctccc aaagtgccaaacctgttttt 124080 tatgtgactt ggtattaaat atttattaaa aagttcaaaa ttgggctcagcacagtagct 124140 cacgcctgta attccagcac tttgggaggc tgaggcaggc agatcgtttaaggtcaggag 124200 tttgagacca gcctgaccaa catggcaaaa ccctgtccct actaaaaatacaaaaaatta 124260 gctgggcatg atcacacatg cctgtaatcc cagctactgg ggaggcagaggcactagaat 124320 cttttgagcc ccagaggtgg aggttgcagt gatctgagat cacgccattgcacttcagcc 124380 tgggtgacag agcaagactt tgtctcaaaa aacaaaacaa aacaaaaagagttcaaaatt 124440 caacagtgta tgtcattgcc ttctctatag ggtaccagtc gtccttcacactatcatgtt 124500 ttatgggatg ataactgctt tactgcagat gaacttcagc tgctaacttaccagctctgc 124560 cacacttacg tacgctgtac acgatctgtt tctatacctg caccagcgtattatgctcac 124620 ctggtagcat ttagagccag atatcatctt gtggacaaag aacatgacaggtaatataaa 124680 agcataacag gttctcaccc aaatcccaat attgtctgca tggtaggattttcaagttcc 124740 acaagctatt agcggagtca gtgatccatg tgaaaaatga tgacagaactgactgcccaa 124800 ggtttcctat tgaaatatat tgtctaggct cattagtaat agaatcatgtagtaacttag 124860 ttgttttact acattaattc aaaggagaga ttattggtaa taaagtgatacattcagaca 124920 gatagacaaa atgatacgat atctaggttt gcttctaatt aattctggggaggtgagaga 124980 agtaaaacaa aattggcctt gagttaatag tgattgaagc tgagtgatggacacatagat 125040 tgttacacca gtctcttatt tgttaataat tttgaaattt tctacaacaaatagtatttt 125100 tcggaattat tacatcagaa tagaaagttt gtttttgtgt tcattgccacttctcttaca 125160 gtgctgaagg aagtcacgtt tcaggacaaa gcaatgggcg agatccacaagctcttgcca 125220 aggctgtaca gattcaccaa gataccttac gcacaatgta cttcgcttaaatagtccaag 125280 tatattctct gagaggaagt actgaaagat gaattgacat acaacgtatgtttccagtga 125340 agtcaattga gtaaggacac ctccagccat acagaaacca acactgtgtgggggccaagg 125400 tctgatcctt atgttaatac aaggaagatt gtttacttca tcaaggaacacagcatcatt 125460 atgcaatatg aaaccagcca actgcttttt gtgcggtctc ctataggaagtatcgcaatt 125520 gttttgtttt catttcttgt agtctaaccc ttttaatgcc tttacctcaagttgcttggc 125580 agcacaacta tctttgcaaa aaaaagtaaa gaaaaagtaa atgatggtttaaaaaataca 125640 caccttcatg aataatcaaa gtgatttttc agaattatgt gtgcaaaaaattaatgtgca 125700 ttcatatatt cttgtaaaag gtgtctgtgt atttttaaaa tatatacatccatacttcat 125760 atgcatatat atctagatct ggattgataa tagatatata tgtgtctgttatatatttta 125820 gagttcattc cattggggaa ttttctttcc cttttattct acccccactaccgcctttat 125880 ttctctattt cccttgcctt catcacctac atttttttcc cagtcctaccagtgacattc 125940 aaatgttgat gtatctggtt cgtttgaata taaaatatgg caaactagaattctactttt 126000 a 126001 14 1726 DNA H. sapiens CDS (280)...(969) 14aggaaggagg gtggccctac cccagcgggc tcggctcggg gcctccgcgg cagttcgggg 60tccttcaccc gccggctcca gtcctgtgcc gttttccgtc cgcgactctt ccggcccaga 120gctttcggag tgcggttgct caggggaagc cgtcgccgcc cccgcctcgg ggccgagtga 180gagtgcccgt cgcgtcgcgc cgcgtcgccc cccgggccgc ctccttgccg ccagtggcgg 240gctccgttct ccctcgaagc actcccccca gctccatga atg gaa atc ggc tcc 294 MetGlu Ile Gly Ser 1 5 gca gga ccc gct ggg gcc cag ccc cta ctc atg gtg cccaga aga cct 342 Ala Gly Pro Ala Gly Ala Gln Pro Leu Leu Met Val Pro ArgArg Pro 10 15 20 ggc tat ggc acc atg ggc aaa ccc att aaa ctg ctg gct aactgt ttt 390 Gly Tyr Gly Thr Met Gly Lys Pro Ile Lys Leu Leu Ala Asn CysPhe 25 30 35 caa gtt gaa atc cca aag att gat gtc tac ctc tat gag gta gatatt 438 Gln Val Glu Ile Pro Lys Ile Asp Val Tyr Leu Tyr Glu Val Asp Ile40 45 50 aaa cca gac aag tgt cct agg aga gtg aac agg gag gtg gtt gac tca486 Lys Pro Asp Lys Cys Pro Arg Arg Val Asn Arg Glu Val Val Asp Ser 5560 65 atg gtt cag cat ttt aaa gta act ata ttt gga gac cgt aga cca gtt534 Met Val Gln His Phe Lys Val Thr Ile Phe Gly Asp Arg Arg Pro Val 7075 80 85 tat gat gga aaa aga agt ctt tac acc gcc aat cca ctt cct gtg gca582 Tyr Asp Gly Lys Arg Ser Leu Tyr Thr Ala Asn Pro Leu Pro Val Ala 9095 100 act aca ggg gta gat tta gac gtt act tta cct ggg gaa ggt gga aaa630 Thr Thr Gly Val Asp Leu Asp Val Thr Leu Pro Gly Glu Gly Gly Lys 105110 115 gat cga cct ttc aag gtg tca atc aaa ttt gtc tct cgg gtg agt tgg678 Asp Arg Pro Phe Lys Val Ser Ile Lys Phe Val Ser Arg Val Ser Trp 120125 130 cac cta ctg cat gaa gta ctg aca gga cgg acc ttg cct gag cca ctg726 His Leu Leu His Glu Val Leu Thr Gly Arg Thr Leu Pro Glu Pro Leu 135140 145 gaa tta gac aag cca atc agc act aac cct gcc cat gcc gtt gat gtg774 Glu Leu Asp Lys Pro Ile Ser Thr Asn Pro Ala His Ala Val Asp Val 150155 160 165 gtg cta cga cat ctg ccc tcc atg aaa tac aca cct gtg ggg cgttca 822 Val Leu Arg His Leu Pro Ser Met Lys Tyr Thr Pro Val Gly Arg Ser170 175 180 ttt ttc tcc gct cca gaa gga tat gac cac cct ctg gga ggg ggcagg 870 Phe Phe Ser Ala Pro Glu Gly Tyr Asp His Pro Leu Gly Gly Gly Arg185 190 195 gaa gtg tgg ttt gga ttc cat cag tct gtt cgg cct gcc atg tggaaa 918 Glu Val Trp Phe Gly Phe His Gln Ser Val Arg Pro Ala Met Trp Lys200 205 210 atg atg ctt aat atc gat gaa aga gac ctc tgg cag cag tgt ggagaa 966 Met Met Leu Asn Ile Asp Glu Arg Asp Leu Trp Gln Gln Cys Gly Glu215 220 225 tag atagaggaga aaaaactaat ctgagaagcc agttaggagg cttttcaatc1019 actagttcag gtaagagatg gtgatggtct aatgtgggat ggagaggaag gattaagctg1079 aaaaaatagg tttaagaacc atgtccagaa aaaaaaaact ttgggtgtag tactggtaca1139 tggaacggat tggaaaaaaa tagaccagag ctttactaga tttttgaaag aattgttttt1199 gcaaataaac tacaagcctg gcatatcagt caaggttcag aacctagaga agcagaacca1259 gtacgaagta tagagagaga gtttatgcaa ttgtaggggc tagctaggca agtctgaaat1319 tttgggggca ggctgtcagg aagggcaggg aaattcaggc atgggctgaa gcagttatct1379 ataagtggaa tttcttgctc tcagggaagc ttcagcccta cttttaagac ctttcaccta1439 attgaatcag gcccatccac attattcagg ataatctcca ttacttaaag tcaacagatt1499 atggatttta atcacatcta caaaatacct tcatagcaac acctagatta gtgtttgatt1559 aaacaaatgg cagctggtag cctagcaagt taacacatta aaaacaacac ctgccatgat1619 ggcttacact tgtaatccca gcactttggg aggccaaagt gggaggatca cttgagatta1679 ggagtttgcg atcaggctga acaacatagt gagacctgat ctctacc 1726 15 20 DNAArtificial Sequence Antisense Oligonucleotide 15 agaacggagc ccgccactgg20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 ccgatttccattcatggagc 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17cttgaaaaca gttagccagc 20 18 20 DNA Artificial Sequence AntisenseOligonucleotide 18 tcatagaggt agacatcaat 20 19 20 DNA ArtificialSequence Antisense Oligonucleotide 19 gtcaaccacc tccctgttca 20 20 20 DNAArtificial Sequence Antisense Oligonucleotide 20 attgagtcaa ccacctccct20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 ctttaaaatgctgaaccatt 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22ccatcataaa ctggtctacg 20 23 20 DNA Artificial Sequence AntisenseOligonucleotide 23 tggcggtgta aagacttctt 20 24 20 DNA ArtificialSequence Antisense Oligonucleotide 24 agtggattgg cggtgtaaag 20 25 20 DNAArtificial Sequence Antisense Oligonucleotide 25 ctgtagttgc cacaggaagt20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 ctaaatctacccctgtagtt 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27gtcctgtcag tacttcatgc 20 28 20 DNA Artificial Sequence AntisenseOligonucleotide 28 ctaattccag tggctcaggc 20 29 20 DNA ArtificialSequence Antisense Oligonucleotide 29 catcaacggc atggacaggg 20 30 20 DNAArtificial Sequence Antisense Oligonucleotide 30 tttcatggag ggcagatgtc20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 aggtgtgtatttcatggagg 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32tctggagcgg agaaaaatga 20 33 20 DNA Artificial Sequence AntisenseOligonucleotide 33 gaacagactg atggaatcca 20 34 20 DNA ArtificialSequence Antisense Oligonucleotide 34 ctcattgttc cacaatgagt 20 35 20 DNAArtificial Sequence Antisense Oligonucleotide 35 gcctccttgt tacattacaa20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 tctctccacagtttggccgt 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37agagtatact tttctctgaa 20 38 20 DNA Artificial Sequence AntisenseOligonucleotide 38 gggtacttca gctgaagagt 20 39 20 DNA ArtificialSequence Antisense Oligonucleotide 39 cctgcccgac ttgcagacag 20 40 20 DNAArtificial Sequence Antisense Oligonucleotide 40 tcttgtctat ctggtgcaga20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 aatttgcacttcttaccaat 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42gatctgtttc ataatttgca 20 43 20 DNA Artificial Sequence AntisenseOligonucleotide 43 tgaaactcct gaacaaatgg 20 44 20 DNA ArtificialSequence Antisense Oligonucleotide 44 ctttaaattg aaactcctga 20 45 20 DNAArtificial Sequence Antisense Oligonucleotide 45 atcccgaact ttaaattgaa20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 ccatttcatcccgaacttta 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47acatgagcca tttcatcccg 20 48 20 DNA Artificial Sequence AntisenseOligonucleotide 48 gaagtacgcg tccagttaca 20 49 20 DNA ArtificialSequence Antisense Oligonucleotide 49 ctgtccgatt ccgtcctcca 20 50 20 DNAArtificial Sequence Antisense Oligonucleotide 50 catactccat ggctcggtgt20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 ttcaactcctgtgtggaatt 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52gtgaaaccct tcaatatttc 20 53 20 DNA Artificial Sequence AntisenseOligonucleotide 53 gccagaatat gtgttcttga 20 54 20 DNA ArtificialSequence Antisense Oligonucleotide 54 gacgataata agctgtaggc 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 ttacattctt gacttgaaca20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 taacatttatctttaggcac 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57aatattattg atccctccga 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 gtggatgagt gacatcggct 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 aagttcccgg accatggagg 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 cctctgaaac accatcccga20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 tagttcataatataatacct 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62ctccaaactg atgcaggctt 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 tacaatgtag gttattccag 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 gcacaaaata atcgagtgtg 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 tccaaccctt tctgtcctat20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 gggatattgccacttcttcc 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67gtgtgtaatg tctgtatcaa 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 ctaccaggtg agcataatac 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 ccttggcaag agcttgtgga 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 ttgtgcgtaa ggtatcttgg20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 ttggactatttaagcgaagt 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72agtacttcct ctcagagaat 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 ggaggtgtcc ttactcaatt 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 taaggatcag accttggccc 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 gatgctgtgt tccttgatga20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 agaccgcacaaaaagcagtt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77caaagatagt tgtgctgcca 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 aggtctcttt catcgatatt 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 gctttcgttc taacaggagg 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 tggtgaataa aatgacaatc20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 cagaaatccaccacccaata 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82ataccacgtt ctgatttccc 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 aaccacctcc ctaaagaagg 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 aggtctcttt ctggaaaaca 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 aagcatagaa actttcagtt20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 gagactttacccgtcctcca 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87accagtcaga ctctctgtgt 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 gaactcctga cctcaggtga 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 gtaggcttac ctgtattcca 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 cggcacagga ctggagccgg20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 tgagcaaccgcactccgaaa 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92ttcccctgag caaccgcact 20 93 20 DNA H. sapiens 93 ccagtggcgg gctccgttct20 94 20 DNA H. sapiens 94 gctccatgaa tggaaatcgg 20 95 20 DNA H. sapiens95 gctggctaac tgttttcaag 20 96 20 DNA H. sapiens 96 attgatgtctacctctatga 20 97 20 DNA H. sapiens 97 tgaacaggga ggtggttgac 20 98 20 DNAH. sapiens 98 agggaggtgg ttgactcaat 20 99 20 DNA H. sapiens 99aatggttcag cattttaaag 20 100 20 DNA H. sapiens 100 cgtagaccag tttatgatgg20 101 20 DNA H. sapiens 101 aagaagtctt tacaccgcca 20 102 20 DNA H.sapiens 102 ctttacaccg ccaatccact 20 103 20 DNA H. sapiens 103acttcctgtg gcaactacag 20 104 20 DNA H. sapiens 104 aactacaggg gtagatttag20 105 20 DNA H. sapiens 105 gcatgaagta ctgacaggac 20 106 20 DNA H.sapiens 106 gcctgagcca ctggaattag 20 107 20 DNA H. sapiens 107ccctgtccat gccgttgatg 20 108 20 DNA H. sapiens 108 gacatctgcc ctccatgaaa20 109 20 DNA H. sapiens 109 cctccatgaa atacacacct 20 110 20 DNA H.sapiens 110 tcatttttct ccgctccaga 20 111 20 DNA H. sapiens 111tggattccat cagtctgttc 20 112 20 DNA H. sapiens 112 actcattgtg gaacaatgag20 113 20 DNA H. sapiens 113 ttgtaatgta acaaggaggc 20 114 20 DNA H.sapiens 114 acggccaaac tgtggagaga 20 115 20 DNA H. sapiens 115ttcagagaaa agtatactct 20 116 20 DNA H. sapiens 116 actcttcagc tgaagtaccc20 117 20 DNA H. sapiens 117 ctgtctgcaa gtcgggcagg 20 118 20 DNA H.sapiens 118 tctgcaccag atagacaaga 20 119 20 DNA H. sapiens 119attggtaaga agtgcaaatt 20 120 20 DNA H. sapiens 120 tgcaaattat gaaacagatc20 121 20 DNA H. sapiens 121 ccatttgttc aggagtttca 20 122 20 DNA H.sapiens 122 tcaggagttt caatttaaag 20 123 20 DNA H. sapiens 123ttcaatttaa agttcgggat 20 124 20 DNA H. sapiens 124 taaagttcgg gatgaaatgg20 125 20 DNA H. sapiens 125 cgggatgaaa tggctcatgt 20 126 20 DNA H.sapiens 126 tgtaactgga cgcgtacttc 20 127 20 DNA H. sapiens 127tggaggacgg aatcggacag 20 128 20 DNA H. sapiens 128 acaccgagcc atggagtatg20 129 20 DNA H. sapiens 129 aattccacac aggagttgaa 20 130 20 DNA H.sapiens 130 gaaatattga agggtttcac 20 131 20 DNA H. sapiens 131tcaagaacac atattctggc 20 132 20 DNA H. sapiens 132 gcctacagct tattatcgtc20 133 20 DNA H. sapiens 133 tgttcaagtc aagaatgtaa 20 134 20 DNA H.sapiens 134 gtgcctaaag ataaatgtta 20 135 20 DNA H. sapiens 135agccgatgtc actcatccac 20 136 20 DNA H. sapiens 136 cctccatggt ccgggaactt20 137 20 DNA H. sapiens 137 tcgggatggt gtttcagagg 20 138 20 DNA H.sapiens 138 aggtattata ttatgaacta 20 139 20 DNA H. sapiens 139aagcctgcat cagtttggag 20 140 20 DNA H. sapiens 140 ctggaataac ctacattgta20 141 20 DNA H. sapiens 141 cacactcgat tattttgtgc 20 142 20 DNA H.sapiens 142 ataggacaga aagggttgga 20 143 20 DNA H. sapiens 143ttgatacaga cattacacac 20 144 20 DNA H. sapiens 144 gtattatgct cacctggtag20 145 20 DNA H. sapiens 145 tccacaagct cttgccaagg 20 146 20 DNA H.sapiens 146 ccaagatacc ttacgcacaa 20 147 20 DNA H. sapiens 147acttcgctta aatagtccaa 20 148 20 DNA H. sapiens 148 attctctgag aggaagtact20 149 20 DNA H. sapiens 149 aattgagtaa ggacacctcc 20 150 20 DNA H.sapiens 150 gggccaaggt ctgatcctta 20 151 20 DNA H. sapiens 151tcatcaagga acacagcatc 20 152 20 DNA H. sapiens 152 aactgctttt tgtgcggtct20 153 20 DNA H. sapiens 153 tggcagcaca actatctttg 20 154 20 DNA H.sapiens 154 cctcctgtta gaacgaaagc 20 155 20 DNA H. sapiens 155gattgtcatt ttattcacca 20 156 20 DNA H. sapiens 156 tattgggtgg tggatttctg20 157 20 DNA H. sapiens 157 gggaaatcag aacgtggtat 20 158 20 DNA H.sapiens 158 ccttctttag ggaggtggtt 20 159 20 DNA H. sapiens 159tgttttccag aaagagacct 20 160 20 DNA H. sapiens 160 tggaggacgg gtaaagtctc20 161 20 DNA H. sapiens 161 acacagagag tctgactggt 20 162 20 DNA H.sapiens 162 tcacctgagg tcaggagttc 20 163 20 DNA H. sapiens 163tttcggagtg cggttgctca 20 164 20 DNA H. sapiens 164 agtgcggttg ctcaggggaa20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding PAZ/PIWI domain-containing protein,wherein said compound specifically hybridizes with said nucleic acidmolecule encoding PAZ/PIWI domain-containing protein and inhibits theexpression of PAZ/PIWI domain-containing protein.
 2. The compound ofclaim 1 which is an antisense oligonucleotide.
 3. The compound of claim2 wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 3 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 5 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 7 wherein the modifiednucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound8 to 80 nucleobases in length which specifically hybridizes with atleast an 8-nucleobase portion of a preferred target region on a nucleicacid molecule encoding PAZ/PIWI domain-containing protein.
 11. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 12. The composition of claim 11 furthercomprising a colloidal dispersion system.
 13. The composition of claim11 wherein the compound is an antisense oligonucleotide.
 14. A method ofinhibiting the expression of PAZ/PIWI domain-containing protein in cellsor tissues comprising contacting said cells or tissues with the compoundof claim 1 so that expression of PAZ/PIWI domain-containing protein isinhibited.
 15. A method of treating an animal having a disease orcondition associated with PAZ/PIWI domain-containing protein comprisingadministering to said animal a therapeutically or prophylacticallyeffective amount of the compound of claim 1 so that expression ofPAZ/PIWI domain-containing protein is inhibited.
 16. The method of claim15 wherein the disease or condition is a hyperproliferative disorder.17. The method of claim 16 wherein the hyperproliferative disorder iscancer.
 18. The method of claim 15 wherein the disease or conditionarises from aberrant cellular differentiation.
 19. A method of screeningfor an antisense compound, the method comprising the steps of: a.contacting a preferred target region of a nucleic acid molecule encodingPAZ/PIWI domain-containing protein with one or more candidate antisensecompounds, said candidate antisense compounds comprising at least an8-nucleobase portion which is complementary to said preferred targetregion, and b. selecting for one or more candidate antisense compoundswhich inhibit the expression of a nucleic acid molecule encodingPAZ/PIWI domain-containing protein.
 20. A method of modulating aRNA-mediated interference (RNAi) pathway in an organism comprisingadministering to said organism a therapeutically or prophylacticallyeffective amount of the compound of claim 1 so that expression of thegene encoding PAZ/PIWI domain-containing protein is inhibited.